Bio-engineered hyper-functional &#34;super&#34; helicases

ABSTRACT

Conformationally-constrained helicases having improved activity and strength are provided. Methods of making conformationally-constrained helicases having improved activity and strength are provided. Methods of using conformationally-constrained helicases having improved activity and strength are provided. The present invention is based on the discovery of novel modified helicases that show dramatically enhanced helicase activity and increased strength as compared to unmodified helicases. As described further herein, it has been surprisingly discovered that, by controlling the conformation of certain subdomains such that the helicase remains in a closed form (e.g., by covalently crosslinking the 2B domain to the 1A domain or the 1B domain in a Rep helicase), a highly active and strong form of the helicase is achieved

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/079,183, filed Nov. 13, 2014, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM065367 awarded by the National institutes of Health. The United States Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods fix helicase-mediated DNA unwinding activity.

BACKGROUND

A traditional definition of a helicase is an enzyme that catalyzes the reaction of separating/unzipping/unwinding the helical structure of nucleic acid duplexes (DNA, RNA or hybrids) into single-stranded components, using nucleoside triphosphate (NTP) hydrolysis as the energy source (such as ATP). However, it should be noted that not all helicases fit this definition anymore. A more general definition is that they are motor proteins that move along the single-stranded or double stranded nucleic acids (usually in a certain direction, 3′ to 5′ or 5 to 3, or both), i.e. translocases, that can or cannot unwind the duplexed nucleic acid encountered. In addition, some helicases simply bind and “melt” the duplexed nucleic acid structure without an apparent translocase activity.

Helicases exist in all living organisms and function in all aspects of nucleic acid metabolism. Helicases are classified based on the amino acid sequences, directionality, oligomerization state and nucleic-acid type and structure preferences. The most common classification method was developed based on the presence of certain amino acid sequences, called motifs. According to this classification helicases are divided into 6 super families: SF1, SF2, SF3, SF4, SF5 and SF6. SF1 and SF2 helicases do not form a ring structure around the nucleic acid, whereas SF3 to SF6 do. Superfamily classification is not dependent on the classical taxonomy.

DNA helicases are responsible for catalyzing the unwinding of double-stranded DNA (dsDNA) molecules to their respective single-stranded nucleic acid (ssDNA) forms. Although structural and biochemical studies have shown how various helicases can translocate on ssDNA directionally, consuming one ATP per nucleotide, the mechanism of nucleic acid unwinding and how the unwinding activity is regulated remains unclear and controversial (T. M. Lohman, E. J. Tomko, C. G. Wu, “Non-hexameric DNA helicases and translocases: mechanisms and regulation,” Nat Rev Mol Cell Biol 9:391-401 (2008)). Since helicases can potentially unwind all nucleic acids encountered, understanding how their unwinding activities are regulated can lead to harnessing helicase functions for biotechnology applications.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the discovery of novel modified helicases that show dramatically enhanced helicase activity and increased strength as compared to unmodified helicases. As described further herein, it has been surprisingly discovered that, by controlling the conformation of certain subdomains such that the helicase remains in a closed form (e.g., by covalently crosslinking the 2B domain to the 1A domain or the 1B domain in a Rep helicase), a highly active and strong form of the helicase is achieved.

In one aspect, a composition for catalyzing an unwinding reaction on double-stranded DNA is provided that includes a conformationally-constrained helicase.

In another aspect, a method of catalyzing an unwinding reaction of a double-stranded DNA is provided. The method includes the step of contacting the double-stranded DNA with a conformationally-constrained helicase in the presence of ATP.

In another aspect, an isolated nucleic acid that encodes a helicase polypeptide having the capability to be constrained in a conformation by an intramolecular crosslinking agent is provided.

In another aspect, a modified helicase comprising a first subdomain having a first amino acid and a second subdomain having a second amino acid is provided. Said first amino acid is at least about 30 Å from said second amino acid when the helicase is in an inactive conformation, and said first amino acid is less than about 20 Å from said second amino acid when the helicase is in an active conformation. A side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained helicase.

In certain exemplary embodiments, the modified helicase is a Super Family 1 (SF1) helicase (e.g., an SF1A or an SF1B helicase) or a Super Family 2 (SF2) helicase.

In certain exemplary embodiments, the first amino acid is less than about 20 Å, about 19 Å, about 18 Å, about 17 Å, about 16 Å, about 15 Å, about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 5 Å, or about 4 Å from the second amino acid when the helicase is in an active conformation.

In certain exemplary embodiments, the first amino acid is at least about 30 Å, about 40 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å or about 85 Å from the second amino acid when the helicase is in an inactive conformation.

In certain exemplary embodiments, the helicase is selected from the group consisting of a Rep helicase (e.g., from E. coli.), a UvrD helicase (e.g., from E. coli.) and a PcrA helicase (e.g., from B. stearothermophilus).

In certain exemplary embodiments, the first amino acid is at any one of positions 84-116 or 178-196 of the modified helicase amino acid sequence, and the helicase is a Rep, PcrA or UvrD helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one of positions 92-116 or 178-196 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one of positions 84-108 or 169-187 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one of positions 90-114 or 175-193 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid at position 178 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at position 187 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:13 or SEQ ID NO:14, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:15 or SEQ ID NO:16, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid residue is at any one of positions 388-411, 422-444 and 518-540 of the modified helicase amino acid sequence, and the helicase is a Rep, PcrA or UvrD helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one of positions 397-411, 431-444 or 526-540 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one of positions 388-402, 422-435 or 519-531 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one of positions 393-407, 427-440 or 523-540 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at position 400 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at position 409 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one of positions 60-82 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof. In certain exemplary embodiments, the first amino acid is at any one of positions 68-79 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one of positions 69-89 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof. In certain exemplary embodiments, the first amino acid is at any one of positions 77-87 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one of positions 67-87 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof. In certain exemplary embodiments, the first amino acid is at any one of positions 75-85 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one of positions 509-536 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof. In certain exemplary embodiments, the second amino acid is at any one of positions 519-525 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one of positions 516-534 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof. In certain exemplary embodiments, the second amino acid is at any one of positions 526-532 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one of positions 513-531 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof. In certain exemplary embodiments, the second amino acid is at any one of positions 523-529 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof.

In certain exemplary embodiments, said first subdomain and said second subdomain comprise no more than a total of two cysteine residues.

In certain exemplary embodiments, the helicase comprises one cysteine residue and/or is from a bacterium selected from the group consisting of Deinococcus geothermalis, Meiothermus sp., Marinithermus hydrothermalis, Marinithermus hydrothermalis and Oceanithermus profundus.

In certain exemplary embodiments, the helicase comprises one cysteine residue or no cysteine residues and/or is from a bacterium selected from the group consisting of Thermococcus sp. EXT9, Thermococcus sp. IRI48 Thermococcus sp. IRI33, Thermococcus sp. AMT7, Thermococcus nautili, Thermococcus onnurineus (strain NA1), Thermococcus kodakarensis (strain ATCC BAA-918/JCM 12380/KOD1) (Pyrococcus kodakaraensis (strain KOD1)), Thermococcus sibiricus (strain MM 739/DSM 12597), Thermococcus paralvinellae, Thermus aquaticus Y51MC23, Thermus aquaticus Y51MC23, Thermus aquaticus Y51MC23, Thermus sp. RL, Thermus sp. RL, Thermus sp. 2.9, Salinisphaera hydrothermalis C41B8, Thermus filiformis, Meiothermus ruber, Thermus sp. NMX2.A1, Thermus thermophilus JL-18, Thermus scotoductus (strain ATCC 700910/SA-01), Thermus scotoductus (strain ATCC 700910/SA-01), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274506), Thermus oshimai JL-2, Thermus oshimai JL-2, Thermus oshimai JL-2, Thermomonospora curvata (strain ATCC 19995/DSM 43183/JCM 3096/NCIMB 10081), Thermodesulfatator indicus (strain DSM 15286/JCM 11887/CIR29812), Geobacillus stearothermophilus (Bacillus stearothermophilus), Coprothermobacter proteolyticus (strain ATCC 35245/DSM 5265/BT), Meiothermus silvanus (strain ATCC 700542/DSM 9946/VI-R2) (Thermus silvanus), Anaerolinea thermophila (strain DSM 14523/JCM 11388/NBRC 100420/UNI-1), Thermoanaerobacterium thermosaccharolyticum M0795, Meiothermus ruber (strain ATCC 35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Meiothermus ruber (strain ATCC 35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Deinococcus radiodurans (strain ATCC 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/NCIMB 9279/R1/VKM B-1422), Thermodesulfobium narugense DSM 14796, Thermus thermophilus (strain HB8/ATCC 27634/DSM 579), Dictyoglomus thermophilum (strain ATCC 35947/DSM 3960/H-6-12), Thermus thermophilus (strain SG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermus sp. CCB_US3_UF1, Deinococcus geothermalis (strain DSM 11300), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM 7039), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM 7039), Marinithermus hydrothermalis (strain DSM 14884/JCM 11576/T1).

In certain exemplary embodiments, the first amino acid and the second amino acid are each independently an unnatural amino acid or a natural amino acid.

In certain exemplary embodiments, one or more of an amino acid of the helicase is substituted with an unnatural amino acid or a natural amino acid (e.g., a cysteine or a homocysteine).

In certain exemplary embodiments, said helicase comprises a sequence selected from SEQ ID NOs:4 and 12.

In certain exemplary embodiments, the first amino acid is covalently crosslinked to the second amino acid by a disulfide bond or by a chemical crosslinker (e.g., a chemical crosslinker having a length of from about 6 Å to about 25 Å).

In certain exemplary embodiments, the chemical crosslinker is a his-maleimide crosslinker.

In certain exemplary embodiments, the chemical crosslinker is selected from the group consisting of

1-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione,

1-[6-(2,5-dioxopyrrol-1-yl)hexyl]pyrrole-2,5-dione,

1-[2-[2-(2,5-dioxopyrrol-1-yl)ethyldisulfanyl]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)phenyl]pyrrole-2,5-dione, and

N,N′-bis[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethyl]-N,N′-diphenylbutanediamide.

In certain exemplary embodiments, the chemical crosslinker is

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione.

In one aspect, a modified helicase comprising a first subdomain having a first amino acid and a second subdomain having a second amino acid, wherein said first amino acid is at least about 30 Å from said second amino acid when the helicase is in an inactive conformation, and said first amino acid is less than about 20 Å from said second amino acid when the helicase is in an active conformation, and wherein a side chain of the first amino acid is chemically crosslinked to a side chain of the second amino acid using

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione to form an active, conformationally-constrained helicase is provided.

In another aspect, a modified Rep, PcrA or UvrD helicase or homolog thereof, comprising a first subdomain having a first amino acid at any one of positions 84-116 and a second subdomain having a second amino acid at any one of positions 388-411, 422-444 and 518-540, wherein a side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained Rep, PcrA or UvrD helicase, or homolog thereof is provided.

In another aspect, a modified Rep helicase or homolog thereof comprising an amino acid at position 178 covalently crosslinked to an amino acid at position 400 to form an active, conformationally-constrained Rep helicase or homolog thereof is provided.

In another aspect, a modified Rep helicase or homolog thereof comprising an amino acid at position 187 covalently crosslinked to an amino acid at position 409, to form an active, conformationally-constrained helicase is provided.

In another aspect, a modified helicase comprising a first subdomain having a first amino acid and a second subdomain having a second amino acid, wherein said first amino acid is at least about 30 Å from said second amino acid when the helicase is in an inactive conformation, and said first amino acid is less than about 20 Å from said second amino acid when the helicase is in an active conformation, and wherein a side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a chemical crosslinker to form an active, conformationally-constrained helicase, and wherein one or more of an amino acid of the helicase is substituted with an unnatural amino acid or a natural amino acid is provided.

In one aspect, a method of making an active, conformationally-constrained helicase is provided. The method includes the steps of selecting in a helicase a first amino acid in a first subdomain that is at least about 30 Å from a second amino acid in a second subdomain when the helicase is in an inactive conformation, and the first amino acid is less than about 20 Å from the second amino acid when the helicase is in an active conformation, and covalently crosslinking the first amino acid to the second amino acid when the helicase is in an active conformation to form an active, conformationally-constrained helicase.

In a certain exemplary embodiment, the method includes two steps. The first step includes expressing a helicase polypeptide having the capability to be constrained in a conformation by an intramolecular crosslinking agent from an isolated nucleic acid selected from a group consisting of SEQ ID NOs: 2, 3, 5 and 6. The second step includes reacting the helicase polypeptide with an intramolecular crosslinking agent to form the conformationally-constrained helicase.

In certain exemplary embodiments, the modified helicase is a Super Family 1 (SF1) helicase (e.g., SF1A or SF1B) or a Super Family 2 (SF2) helicase.

In certain exemplary embodiments, the first subdomain comprises a 1A subdomain or a 1B subdomain and the second subdomain comprises a 2B subdomain.

In certain exemplary embodiments, the first amino acid is less than about 20 Å, about 19 Å, about 18 Å, about 17 Å, about 16 Å, about 15 Å, about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 5 Å, or about 4 Å from the second amino acid when the helicase is in an active conformation.

In certain exemplary embodiments, the first amino acid is at least about 30 Å, about 35 Å, about 40 Å, about 45 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å or about 85 Å from the second amino acid when the helicase is in an inactive conformation.

In certain exemplary embodiments, the helicase is selected from the group consisting of a Rep helicase, a UvrD helicase and a PcrA helicase.

In certain exemplary embodiments, the helicase comprises a sequence selected from SEQ ID NOs:4 and 12.

In certain exemplary embodiments, the first amino acid is covalently linked to the second amino acid by a disulfide bond or a chemical crosslinker.

In another aspect, a method of catalyzing an unwinding reaction of a double-stranded DNA, comprising contacting the double-stranded DNA with a modified helicase comprising a first subdomain having a first amino acid and a second subdomain having a second amino acid is provided. Said first amino acid is at least about 30 Å from said second amino acid when the helicase is in an inactive conformation, and said first amino acid is less than about 20 Å from said second amino acid when the helicase is in an active conformation. A side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained helicase.

In certain exemplary embodiments, the conformationally-constrained helicase comprises SEQ ID NO: 4 or SEQ ID NO:12.

In certain exemplary embodiments, the conformationally-constrained helicase is chemically crosslinked.

In certain exemplary embodiments, the linker comprises an alkyl having a length in the range from C7 to C23 or from C8 to C13.

In another aspect, a method of performing isothermal DNA amplification, comprising combining a DNA template, the conformationally-constrained helicase described above and amplification reagents. under conditions compatible for performing isothermal DNA amplification.

In certain exemplary embodiments, the method includes two steps. The first step includes forming a mixture. The mixture includes a double-stranded DNA template having a first strand and a second strand; a conformationally-constrained helicase; a DNA-dependent DNA polymerase; a first oligonucleotide primer complementary to a portion of the first strand; a second oligonucleotide primer complementary to a portion of the second strand; and an amplification buffer cocktail. The second step includes incubating the mixture at a temperature compatible for activating the conformationally-constrained helicase and DNA-dependent DNA polymerase.

In certain exemplary embodiments, the conformationally-constrained helicase comprises SEQ ID NO:4 or 12. In certain exemplary embodiments, the DNA-dependent DNA polymerase is selected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol Large Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment, Bsu DNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase and phi29 DNA Polymerase.

In certain exemplary embodiments, the conformationally-constrained helicase is chemically crosslinked.

In certain exemplary embodiments, the chemical crosslinker comprises a length in the range from about 6 Å to about 25 Å.

In certain exemplary embodiments, the chemical crosslinker comprises an alkyl having a length in the range from C7 to C23 or from C8 to C13.

In another aspect, a kit for performing helicase dependent amplification is provided. The kit includes a conformationally-constrained helicase and amplification reagents (e.g., an amplification buffer cocktail).

In certain exemplary embodiments, the conformationally-constrained helicase is selected from SEQ ID NOs: 4 and 12.

In certain exemplary embodiments, the kit further comprising a DNA-dependent DNA polymerase, e.g., selected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol I Large Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment, Bsu DNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase and phi29 DNA Polymerase.

In one aspect, an isolated nucleic acid encoding a modified helicase described herein is provided.

In certain exemplary embodiments, the isolated nucleic acid is selected from the group consisting of SEQ ID NOs: 2, 3, 10 and 11.

In one aspect, a modified E. coli. Rep helicase comprising a first subdomain having a first amino acid, a second subdomain having a second amino acid, and an axis vector defined by the alpha carbon of ILE371 from which the vector originates and the alpha carbon of SER280 or the alpha carbon of ALA603, wherein theta is an angle of rotation of said first amino acid and said second amino acid around the axis vector is provided. A first theta between said first amino acid and said second amino acid is between about 60 degrees and about 155 degrees when the helicase is in an inactive conformation, and a second theta between said first amino acid and said second amino acid is between about 355 degrees and about 25 degrees when the helicase is in an active conformation. A side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained helicase.

In certain exemplary embodiments, the first theta is about 133 degrees and/or the second theta is about 0 degrees.

In certain exemplary embodiments, the axis vector is defined by the alpha carbon of ILE371 and the alpha carbon of SER280.

In certain exemplary embodiments, the first amino acid is at any one of positions 84-108 or 169-187 or at position 178 of the modified helicase amino acid sequence. In certain exemplary embodiments, the first amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:13 or SEQ ID NO:14. In certain exemplary embodiments, the first amino acid is at any one of positions 60-82 of the modified helicase amino acid sequence. In certain exemplary embodiments, the first amino acid is at any one of positions 68-79 of the modified helicase amino acid sequence.

In certain exemplary embodiments, the second amino acid is at any one of positions 388-402, 422-435 or 519-531 or at position 400 of the modified helicase amino acid sequence. In certain exemplary embodiments, the first amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:15 or SEQ ID NO:16. In certain exemplary embodiments, the second amino acid is at any one of positions 509-536 of the modified helicase amino acid sequence. In certain exemplary embodiments, the second amino acid is at any one of positions 519-525 of the modified helicase amino acid sequence.

These and other features, objects and advantages of the present invention will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of the invention.

BRIEF DESCRIPTION OF TILE DRAWINGS

The foregoing and other features and advantages of the present invention will be more filly understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A depicts the closed form Rep crystal structure (PDB entry 1UAA), wherein subdomains are colored and named accordingly and 3′ end of the ssDNA (gray) is visible. Residues that were mutated to cysteine and crosslinked to lock the conformation are shown as pink, blue and red van der Waals spheres in both conformations as reference. Boxed area is magnified view showing the two residues that were crosslinked for engineering Rep-X.

FIG. 1B depicts the open form Rep crystal structure (PDB entry 1UAA), wherein subdomains are colored and named accordingly and 3′ end of the ssDNA (gray) is visible. Residues that were mutated to cysteine and crosslinked to lock the conformation are shown as pink, blue and red van der Waals spheres in both conformations as reference. Boxed area is magnified view showing the two residues that were crosslinked for engineering Rep-Y.

FIG. 1C depicts a schematic showing that helicase-catalyzed unwinding of a DNA labeled with a donor and an acceptor would convert high FRET efficiency (E_(FRET)) to low E_(FRET). Shading level of the donor and acceptor color represents the relative intensity changes. Figure discloses “(dT)₁₀” as SEQ ID NO: 33.

FIG. 10 depicts an ensemble unwinding kinetics of DNA from FIG. 1C by Rep and Rep-X shows the enhanced helicase activity of Rep-X over Rep as measured via ensemble E_(FRET). Solid lines are fitted exponential decay curves as guides to the eye.

FIG. 1E depicts exemplary data of ensemble unwinding kinetics of the Rep-Y, Rep-X and non-crosslinked Rep using an assay containing 10 nM helicase, 5 nM 50-bp ensemble unwinding DNA with 3′-(dT)₃₀ (SEQ ID NO: 17) overhang in buffer D and 1 mM ATP).

FIG. 2A depicts a schematic of unwinding stages of dual labeled DNA by a Rep-X monomer. Color lightness of the donor (green) and acceptor (red) on the DNA represents the change in the emission intensities as the unwinding progresses.

FIG. 2B depicts representative single molecule time traces show the DNA binding, unwinding and dissociation for the acceptor strand for Rep-X, wherein the donor fluorescence signal is in green, acceptor in red and E_(FRET) in blue.

FIG. 2C depicts representative single molecule time traces showing the DNA binding, unwinding and dissociation for the donor strand for Rep-X, wherein the donor fluorescence signal is in green, acceptor in red and E_(FRET) in blue. Unwinding period is denoted by Δt.

FIG. 2D depicts representative single molecule time traces showing the DNA binding and dissociation behavior for the donor strand for Rep, wherein the donor fluorescence signal is in green, acceptor in red and E_(FRET) in blue.

FIG. 2E depicts representative single molecule time traces showing the DNA binding and dissociation behavior for the donor su and for Rep-Y, wherein the donor fluorescence signal is in green, acceptor in red and E_(FRET) in blue.

FIG. 2F depicts a representative distribution of Rep-X unwinding period Δt.

FIG. 2G depicts fractions of DNA binding events that led to unwinding (i.e. exhibited an E_(FRET) increase phase) in smFRET experiments for Rep, Rep-Y and Rep-X. Error bars represent 95% confidence bounds.

FIG. 3A depicts a schematic of the optical tweezers assay depicts a Rep-X molecule tethered to the bead surface that just loaded on the free ssDNA overhang and started to unwind the 6-kbp DNA=.

FIG. 3B depicts unwinding traces showing the extent of processive unwinding by Rep-X on the 6-kbp DNA (colored according to conditions of overhang length, SSB and force, and offset for clarity). Background is color coordinated with the inset to show the two laminar flows.

FIG. 3C depicts an exemplary distribution of Rep-X unwinding velocities (N=38). Mean velocity of unwinding and the standard deviation for each molecule were plotted above (colors as in B). Figure discloses “(dT)₁₀,” “(dT)₁₅” and “(dT)₇₅” as SEQ ID NOS 33-35, respectively.

FIG. 3D depicts exemplary data comparing the fraction of the complete DNA binding events for Rep, Rep-Y and Rep-X. Error bars represent the 95% confidence bounds.

FIG. 3E depicts unwinding by five representative Rep-X molecules in the fixed trap assay are plotted. Pulling force increases during unwinding as the Rep-X pulls the beads closer. Tether breaks appear as sudden force drops.

FIG. 3F depicts exemplary data showing the average of normalized unwinding velocities of 58 Rep-X molecules plotted against the pulling force that shows the high force tolerance of the engineered super-helicase Rep-X. Error bars represent standard error of the mean.

FIG. 4A illustrates a consensus sequence alignment of TxGx motif for 27 organisms within 10 out 11 families, wherein Cys is present at position 96. Leuconostocaceae family species have an alanine at this position. Figure discloses SEQ ID NOS 109-142, respectively, in order of appearance.

FIG. 4B illustrates a consensus sequence alignment of motif III for 27 organisms within 10 families, wherein Cys is present at position 247. Leuconostocaceae family species have an alanine at this position. Figure discloses SEQ ID NOS 143-176, respectively, in order of appearance.

FIG. 5A depicts exemplary ATPase activity of mutant PcrA before (“PcrA”) and after crosslinking (“PcrA-X”). Error bars represent standard deviation over multiple preparations.

FIG. 5B depicts exemplary data of an ensemble unwinding assay for PcrA-X and wild type PcrA. Solid lines are fitted exponential decay curves as visual guides.

FIG. 6A depicts representative single molecule time traces for DNA binding and unwinding by PcrA-X monomers.

FIG. 6B depicts representative single molecule time traces for DNA binding and unwinding by t PcrA monomers, which are incapable of DNA unwinding.

FIG. 6C depicts exemplary data of fractions of enzyme-DNA binding events that led to an unwinding phase fix PcrA and PcrA-X in the smFRET assay. Error bars represent the 95% confidence bounds

FIG. 6D depicts exemplary data showing processive unwinding of 6-kbp DNA by four representative PcrA-X molecules in the optical tweezers assay. Figure discloses “(dT)₁₅” and “(dT)₇₅” as SEQ ID NOS 34 and 35, respectively.

FIG. 6E depicts exemplary data for fractions of enzyme-DNA binding that led to the unwinding of 6-kbp DNA in the optical tweezers assay. Error bars represent the 95% confidence bounds

FIG. 6F depicts a schematic (in subpanel (i)) of the conformational effect of RepD, a stimulatory partner of PcrA, on PcrA as measured in a smFRET assay and E_(FRET) histograms (sub-panel (ii)) showing that the PcrA bound to RepD adduct is biased toward the closed form (high E_(FRET) population) compared to PcrA bound to the bare ori-D DNA.

FIG. 7A shows an exemplary SDS-PAGE analysis of Rep-Y intra-crosslinking, wherein the typical three-band pattern on SDS polyacrylamide gels is evident. Rep-X intra-crosslinking pattern is shown for comparison, wherein the dominant middle band is slightly shifted for Rep-X compared with the corresponding band for Rep-Y. Lane designated as Rep is non-crosslinked Rep.

FIG. 7B shows an exemplary SDS-PAGE analysis of Rep-Y intra-crosslinking in comparison to uncrosslinked Rep (“Rep”). Lane denoted as Rep-Y* depicts β-ME reduced Rep-Y (crosslinked with a di-sulfide crosslinker DTME).

FIG. 7C shows an exemplary size exclusion chromatography (SEC) elution profile for Rep (dotted line) and the Rep-Y sample (solid line).

FIG. 7D shows an exemplary SDS-PAGE analysis of Rep-Y fractions, F1-F7, collected from SEC (FIG. 5C) in comparison with Rep-Y.

FIG. 7E depicts exemplary data of ssDNA dependent ATPase levels of Rep-Y and Rep. Error bars represent standard deviation over multiple preparations.

FIG. 8 depicts a schematic of an isothermal DNA amplification process called helicase dependent amplification, wherein in step 1: DNA helicase (104) contacts a double-stranded DNA (101) to unwind the first and second single strands (102 and 103) and first and second oligonucleotide primers (105 and 106) hybridize to the first and second single strands (102 and 103) respectively; in step 2: DNA-dependent DNA polymerases (107) bind to the 3′-termini of the first and second oligonucleotide primers (105 and 106) to initiate chain elongation of new strands (108 and 109); and in step 3: continued DNA polymerization results in DNA amplification and formation of new double-stranded DNA (110 and 111).

FIG. 9A shows target residues in Rep (SEQ ID NO: 32), PcrA (SEQ ID NO: 177) and UvrD (SEQ ID NO: 178), for −X form crosslinking, calculated based on the criteria and crystal structures in open (inactive) and closed (active) conformations. One residue is chosen from 1A or 1B domain (shaded), and another from 2B (shaded).

FIG. 9B shows 56 representative Rep homologs/orthologs with 90% identity to and 80% overlap, and the corresponding region of domain 1A where crosslinking residues can be chosen. FIGS. 9B-9C disclose SEQ ID NOS 179-235, respectively, in order of appearance.

FIG. 9C shows 56 representative Rep homologs/orthologs with 90% identity to and 80% overlap, and the corresponding region of domain 1B where crosslinking residues can be chosen.

FIG. 9D shows 56 representative Rep homologs/orthologs with 90% identity to and 80% overlap, and the corresponding region of domain 2B where crosslinking residues can be chosen. FIGS. 9D-9F disclose SEQ ID NOS 236-292, respectively, in order of appearance.

FIG. 9E shows 56 representative Rep homologs/orthologs with 90% identity to and 80% overlap, and the corresponding region of domain 2B where crosslinking residues can be chosen in addition to those shown in FIG. 9D.

FIG. 9F shows 56 representative Rep homologs/orthologs with 90% identity to and 80% overlap, and the corresponding region of domain 2B where crosslinking residues can be chosen in addition to those shown in FIG. 9E.

FIG. 9G shows target residues in drUvrD, Rep, PcrA and UvrD, for −X form crosslinking, calculated based on the criteria and crystal structures in open (inactive) and closed (active) conformations. One residue is chosen from 1A or 1B domain (shaded), and another from 2B (shaded). Figure discloses SEQ ID NOS 293-304, respectively, in order of appearance.

FIG. 10 shows the reaction of maleimide-activated compounds to sulfhydryl-bearing compounds.

FIG. 11 shows a closed form crystal structure of D. radiodurans UvrD (drUvrD; Q9RTI9) with target crosslinking regions of domains 1A, 1B and 2B indicated by arrows.

FIG. 12 shows selected target residue pairs for crosslinking, and the specific distances between the pairs, in a ribbon diagram of a structure of RecD2.

FIG. 13 is a ribbon diagram of a CsRecQ/DNA crystal structure.

FIG. 14 shows a schematic diagram of RecQ DNA helicase, and an overlay of RecQ structures which highlight the mobility of the WH domain.

FIG. 15 shows alternate ribbon diagrams of a RecQ1 crystal structure.

FIG. 16 shows a stereo view of a ribbon diagram of a 5′-3′ SF1 superhelicase (T4 Dda).

FIG. 17 shows Rep helicase's 2B domain structure in two different orientations that differ through a rotation around an axis coming out of the plane of the paper. 2B domain orientation can be described by the rotation angle θ with respect to the closed form. θ=0 when the helicase is in the closed form, and θ is 133 degrees when the 2B rotates to the open form.

DETAILED DESCRIPTION

The present disclosure provides details of the discovery of robust enzymes of the superfamily 1 helicases. The helicase enzymes are engineered as crosslinked, conformationally-constrained monomeric configurations providing enhanced unwinding activity on dsDNA substrates. The “super” helicases display inherently strong physical properties having superior characteristics to all presently known natural helicases. The disclosed helicases have utility in isothermal PCR and helicase-dependent amplification processes, as well as in next generation sequencing applications, including nanopore sequencing methods and the like.

Terminology and Definitions

The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. With respect to the use of plural and/or singular toms herein, those having skill in the art can translate from the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for the sake of clarity.

Terms used herein are intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).

Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into sub-ranges.

A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 1-6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

The present invention provides modified helicases that have enhanced enzymatic activity. As used herein, a “helicase” refers to a class of enzymes that function as motor proteins which move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e., DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis. Helicases are often used to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases. They also function to remove nucleic acid-associated proteins and catalyze homologous DNA recombination. Metabolic processes of RNA such as translation, transcription, ribosome biogenesis, RNA splicing, RNA transport, RNA editing, and RNA degradation are all facilitated by helicases. Helicases move incrementally along one nucleic acid strand of the duplex with a directionality and processivity specific to each particular enzyme.

Six super families of helicases are known in the art that are classified based on their shared sequence motifs. Helicases not forming a ring structure are classified in Super Families 1 (SF1) and 2 (SF2). Ring-forming helicases form Super Families 3 (SF3), 4 (SF4), 5 (SF5) and 6 (SF6).

SF1 is further subdivided into SF1A and SF1B helicases. In this group, helicases can have either 3′-5′ (SF1A subfamily) or 5′-3′(SF1B subfamily) translocation polarity. SF1A helicases include, but are not limited to are Rep and UvrD in gram-negative bacteria and PcrA helicase from gram-positive bacteria. SF1B helicases include, but are not limited to RecD and Dda helicases.

SF2 is the largest family of helicases, which are involved in varied cellular processes. They are characterized by the presence of nine conserved motifs: Q, I, Ia, Ib, and II through VI. This family primarily comprises DEAD-box RNA helicases (“DEAD” disclosed as SEQ ID NO: 18). Other helicases in SF2 family are the RecQ-like family and Snf2-like enzymes. Most of the SF2 helicases are type A, with a few exceptions such as the XPD family.

SF3 comprises helicases encoded mainly by small DNA viruses and some large nucleocytoplasmic DNA viruses. They have a 3′-5′ translocation directionality (therefore they are all type A helicases). SF3 helicase include viral helicases such as the papilloma virus E1 helicase.

SF4 helicases have a type B polarity (5′-3′), and function in bacterial or bacteriophage DNA replication. Gp4 from bacteriophage T7 is an SF4 helicase.

SF5 helicases have a type B polarity (5′-3′), and include only the bacterial termination factors Rho.

SF6 helicases contain the core AAA+ that is not included in the SF3 classification. SF6 helicases include, but are not limited to, Mini Chromosome Maintenance (MCM), RuvB, RuvA, and RuvC.

Exemplary helicases according to the invention include, but are not limited to RecD, Upfl, PcrA, Rep, UvrD, Hel308, Mtr4, XPD, NS3, Mssl 16, Prp43, RecG, RecQ, TIR, RapA, Hef, RecB, Pif1, Dna2, Dda, Ul5, RecD2, Tral, Sen1p, SETX, IBP160, ZNFX1, Upf1p, UPF1, Hcs1p, IGHMBP2, Dna2p, DNA2, Mtt1p, MOV10, MOV10L1, HELZ, PR285, ptMRDFL1 and the like.

In certain embodiments of the invention, a helicase comprises subdomains. For example, SF1 helicases comprise subdomains 1A, 1B, 2A and 2B. The 2B subdomain has been shown to rotate between an open conformation and a closed conformation.

As used herein, an “open conformation” refers to the inactive conformation of a helicase in which minimal or no helicase activity occurs. As used herein, a “closed conformation” refers to the active form of a helicase which has helicase activity. Crystal structures depicting the open and/or closed conformations of many helicases have been published in the art.

As described further herein, it has been discovered that, by stabilizing the active (i.e., closed) conformation and destabilizing the inactive (i.e., open) conformation, a modified helicase can be obtained having greatly enhanced helicase activity and strength relative to the corresponding unmodified helicase. According to certain embodiments of the invention, a modified helicase that stabilizes the active (i.e., closed) conformation and destabilizes the inactive (i.e., open) conformation can be generated by covalently linking one or more amino acids in the 2B subdomain to one or more amino acids in the 1A and/or the 1B domain of the helicase. Such a modified helicase is referred to herein as an “active, conformationally constrained helicase” or a “helicase-_(X) polypeptide.” Exemplary helicase-_(X) polypeptides include, but are not limited to, Rep-_(X), PcrA-_(X) and UvrD-_(X). In certain embodiments, a helicase-_(X) polypeptide forms a loop around a target nucleic acid sequence (e.g., a DNA sequence) In other embodiments, a helicase-_(X) polypeptide does not form a loop around a target nucleic acid sequence (e.g., a DNA sequence).

In other embodiments, a helicase is provided that is stabilized in its inactive (i.e., open) conformation and destabilized in its active (i.e., closed) conformation. Such a helicase is referred to as an “inactive, conformationally constrained helicase” or a “helicase-_(Y) polypeptide.” Helicase-_(Y) polypeptides exhibit little or no helicase activity.

In certain embodiments, a helicase-_(X) polypeptide has an increased nucleic acid (e.g., DNA) unwinding activity relative to a corresponding unmodified helicase. In certain aspects, the number of base pairs that can be unwound by a helicase-_(X) polypeptide is increased by about 1000%, about 10,000%, about 100,000% or more (or any ranges or points within the ranges) relative to a corresponding unmodified helicase.

In certain embodiments, a helicase-_(X) polypeptide can unwind at least about 500 base pairs, about 1000 base pairs, about 1500 base pairs, about 2000 base pairs, about 2500 base pairs, about 3000 base pairs, about 3500 base pairs, about 4000 base pairs, about 4500 base pairs, about 5000 base pairs, about 5500 base pairs, about 6000 base pairs, about 6500 base pairs, about 7000 base pairs, about 7500 base pairs, about 8000 base pairs, about 8500 base pairs, about 9000 base pairs, about 9500 base pairs, about 10,000 base pairs or more (or any ranges or points within the ranges) without dissociating from the nucleic acid sequence (e.g., DNA).

In certain embodiments, a helicase-_(X) polypeptide is stronger that the corresponding unmodified helicase, withstanding opposing forces of at least about 10 pN, about 15 pN, about 20 pN, about 25 pN, about 30 pN, about 35 pN, about 40 pN, about 45 pN, about 50 pN, about 55 _(pN,) about 60 pN, or more (or any ranges or points within the ranges).

In certain embodiments, a helicase-_(X) polypeptide comprises a first subdomain comprising a first amino acid and a second subdomain comprising a second amino acid, wherein the first amino acid is at least about 35 Å from the second amino acid when the helicase is in an inactive conformation, and wherein the first amino acid is less than about 25 Å from the second amino acid when the helicase is in an active conformation. In certain embodiments, the first amino acid is at least about 40 Å, about 45 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å, about 85 Å, or more from the second amino acid (or any ranges or points within these ranges) when the helicase is in an inactive (i.e., open) conformation. In certain embodiments, the first amino acid is at most about 20 Å, about 15 Å, about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 6 Å, about 5 Å, about 4 Å, or less from the second amino acid (or any ranges or points within the ranges) when the helicase is in an active (i.e., closed) conformation. In certain embodiments, the linker in a helicase_(X) polypeptide has a length in the range from about 6 Å to about 25 Å.

In certain embodiments, the first amino acid of a helicase-_(X) polypeptide is present in a 1A or a 1B subdomain and the second amino acid of a helicase_(X) polypeptide is present in a 2B subdomain.

In certain embodiments, the Rep-_(X) polypeptide forms a loop around the target nucleic acid (e.g., DNA) sequence. In certain embodiments, the first amino acid of a Rep-_(X) polypeptide that forms a loop is at any one of positions 84-108 or 169-187, or at position 178 of the Rep amino acid sequence. In certain embodiments, the second amino acid of a Rep_(X) polypeptide that forms a loop is at any one of positions 388-402, 422-435 or 519-536, or at position 400 of the Rep amino acid sequence.

In certain embodiments, the PcrA-_(X) polypeptide forms a loop around the target nucleic acid (e.g., DNA) sequence. In certain embodiments, the first amino acid of a PcrA-_(X) polypeptide that forms a loop is at any one of positions 92-116 or 178-196, or at position 187 of the PcrA amino acid sequence. In certain embodiments, the second amino acid of a PcrA-_(X) polypeptide that forms a loop is at any one of positions 397-411, 431-444 or 526-540, or at position 409 of the PcrA amino acid sequence.

In certain embodiments, the UvrD-_(X) polypeptide forms a loop around the target nucleic acid (e.g., DNA) sequence. In certain embodiments, the first amino acid of a UvrD-_(X) polypeptide that forms a loop is at any one of positions 90-114 or 175-193 of the UvrD amino acid sequence. In certain embodiments, the second amino acid of a UvrD-_(X) polypeptide that forms a loop is at any one of positions 393-407, 427-440 or 523-540 of the UvrD amino acid sequence.

In certain embodiments, the Rep-_(X) polypeptide does not form a loop around the target nucleic acid (e.g., DNA) sequence. In certain embodiments, the first amino acid of the Rep-_(X) polypeptide that does not form a loop is at any one of positions 60-82 (i.e., at any one of AREMKERVGQTLGRKEARGLMIS (SEQ ID NO: 19)), or at any one of positions 68-79 (i.e., at any one of GQTLGRKEARGL (SEQ ID NO: 20)) of the Rep amino acid sequence. In certain embodiments, the second amino acid of the Rep-_(X) polypeptide that does not form a loop is at any one of positions 509-536 (i.e., at any one of FSWMTEMLEGSELDEPMTLTQVVTRFTL (SEQ ID NO: 21)), or at any one of positions 519-525 (i.e., at any one of SELDEPM (SEQ ID NO: 22)) of the Rep amino acid sequence.

In certain embodiments, the PcrA-_(X) polypeptide does not form a loop around the target nucleic acid (e.g., DNA) sequence. In certain embodiments, the first amino acid of the PcrA-_(X) polypeptide that does not form a loop is at any one of positions 69-89 (i.e., at any one of AREMRERVQSLLGGAAEDVWI (SEQ ID NO: 23)), or at any one of positions 77-87 (i.e., at any one of QSLLGGAAEDV (SEQ ID NO: 24)) of the PcrA amino acid sequence. In certain embodiments, the second amino acid of the PcrA-_(X) polypeptide that does not form a loop is at any one of positions 516-534 (i.e., at any one of LSVTKHFENVSDDKSLIAF (SEQ ID NO: 25)), or at any one of positions 526-532 (i.e., at any one of SDDKSLI (SEQ ID NO: 26)) of the PcrA amino acid sequence.

In certain embodiments, the UvrD-_(X) polypeptide does not form a loop around the target nucleic acid (e.g., DNA) sequence. In certain embodiments, the first amino acid of the UvrD-_(X) polypeptide that does not form a loop is at any one of positions 67-87 (i.e., at any one of AAEMRHRIGQLMGTSQGGMWV (SEQ ID NO: 27)), or at any one of positions 75-85 (i.e., at any one of GQLMGTSQGGM (SEQ ID NO: 28)) of the UvrD amino acid sequence. In certain embodiments, the second amino acid of the UvrD-_(X) polypeptide that does not form a loop is at any one of positions 513-531 (i.e., at any one of VTATRQFSYNEEDEDLMPL (SEQ ID NO: 29)), or at any one of positions 523-529 (i.e., at any one of EEDEDLM (SEQ ID NO: 30)) of the UvrD amino acid sequence.

In certain embodiments, the first amino acid and/or the second amino acid of a helicase-_(X) polypeptide is present in a particular amino acid sequence having at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45 about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more sequence identity to that of a reference sequence (e.g, a Rep helicase, A PcrA helicase, a UvrD helicase, or a homolog or ortholog thereof).

In certain embodiments, the first amino acid is present in a Rep helicase at an amino acid sequence having at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more amino acid sequence identity (or any ranges or points within the ranges) to FHTLGLDIIKREYAALGMKANFSLF (SEQ ID NO:13). In certain embodiments, the first amino acid is present in a Rep helicase at an amino acid sequence having at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or more amino acid sequence identity (or any ranges or points within the ranges) to GLYDAHLKACNVLDFDDLI (SEQ ID NO:14).

In certain embodiments, the second amino acid is present in a Rep helicase at an amino acid sequence having at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% amino acid sequence identity (or any ranges or points within the ranges) to AYLRVLTNPDDDSAF (SEQ ID NO:15). In certain embodiments, the second amino acid is present in a Rep helicase at an amino acid sequence having at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40% about 45%, about 50% about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92% about 93% about 94%, about 95%, about 96%, about 97%, about 98% or about 99% amino acid sequence identity (or any ranges or points within the ranges) to GEWAMTRNKSMFTA (SEQ ID NO:16).

Suitable amino acid positions for modifying to engineer helicase-_(X) polypeptides (and homologs and orthologs thereof) according to the invention can be identified by one of ordinary skill in the art using this disclosure and well-known local sequence alignment tools.

Techniques fbr determining nucleic acid and amino acid “sequence identity” are known in the art. Typically, such techniques include determining the nucleotide sequence of genomic DNA, mRNA or cDNA made from an mRNA for a gene and/or determining the amino acid sequence that it encodes, and comparing one or both of these sequences to a second nucleotide or amino acid sequence, as appropriate. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.

An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986) Nucl. Acids Res. 14:6745. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.).

One method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “match” value reflects “sequence identity.” Other suitable programs fbr calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by .dbd.HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the NCBI/NLM web site.

In certain embodiments of the invention, a helicase is provided that is conformationally-constrained. The term “conformationally-constrained” refers to a conformation having a least one degree of freedom (that is, motion or range of motion) that is less than a reference conformation. In certain embodiments, a conformationally-constrained helicase has a least one degree of freedom that is less than a helicase that is not conformationally constrained.

In certain embodiments of the invention, a helicase is constrained via a covalent linkage between two or more amino acids of the helicase. A covalent linkage is a chemical linkage between two atoms or radicals formed by the sharing of a pair of electrons (i.e., a single bond), two pairs of electrons (i.e., a double bond) or three pairs of electrons (i.e., a triple bond). Covalent linkages are also known in the art as election pair interactions or electron pair bonds.

In certain embodiments, a covalent linkage is formed via a crosslink between the side chains of two (or more) amino acids of a polypeptide (e.g., between two (or more) amino acids of a modified helicase).

As used herein the term “crosslink” refers to the joining of two or more molecules by a covalent bond. Crosslinking can occur via disulfide bonds, e.g., between cysteine residues. Crosslinking can occur via the use of crosslinking reagents (or chemical crosslinkers), which are molecules that contain two or more reactive ends capable of chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules.

The terms “intramolecular crosslinking agent” and “chemical crosslinking agent” refer to a compound that can form covalent bonds via specific functional groups (e.g., primary amines, sulfhvdryls, etc.) on proteins or other molecules. An example of an intramolecular or chemical crosslinking agent includes a compound having two bifunctional groups in its structure.

Chemical crosslinkers are known in the art, and are commercially available (e.g., from Thermo Fisher Scientific, Waltham, Mass.). In certain embodiments, a crosslinker is cleavable (e.g., by reducing one or more of the functional groups of the crosslinker). In other embodiments, a crosslinker is not cleavable.

Examples of chemical crosslinkers include, but are not limited to, those having the following functional groups: maleimide, active esters, succinimide, azide, alkyne (such as dibenzocyclooctynol (DIBO or DBCO), difluoro cycloalkynes and linear alkynes), phosphine (such as those used in traceless and non-traceless Staudinger ligations), haloacetyl (such as iodoacetamide), phosgene type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazines, disulphides, vinyl sulfones, aziridines and photoreactive reagents (such as aryl azides, diaziridines). Reactions between amino acids and functional goups may be spontaneous, such as cysteine/maleimide, or may require external reagents, such as Cu(I) for linking azide and linear alkynes.

Linkers can comprise any molecule that stretches across the distance required. Linkers can vary in length from one carbon (phosgene-type linkers) to many Angstroms. In certain embodiments, the linker includes an alkyl having a length in the range from C₇ to C₂₃. In some embodiments, the linker includes an alkyl having a length in the range from C₈ to C₁₃.

Examples of linear molecules include but are not limited to, polyethyleneglycols (PEGs), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, and polyamides. These linkers may be inert or reactive, in particular they may be chemically cleavable at a defined position, or may be themselves modified with a ligand. In certain embodiments, the linker is resistant to dithiothreitol (DTT).

Examples of crosslinkers include, but are not limited to 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate, di-maleimide PEG lk, di-maleimide PEG 3.4k, di-maleimide PEG 5k, di-maleimide PEG 10k, bis(maleimido)ethane (BMOE), bis-maleimidohexane (BMH), 1,4-bis-maleimidobutane (BMB), 1,4 bis-maleimidyl-2,3-dihydroxybutane (BMDB), BM[PEO]2 (1,8-bis-maleimidodiethyleneglycol), BM[PEO]3 (1,11-bis-maleimidotriethylene glycol), tris[2-maleimidoethyl]amine (TMFA), dithiobismaleimidoethane (DTME), bis-maleimide PEG3, bis-maleimide PEGU, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8 kDa, DBCO-PEG-DBCO 4.0 kDa, DBCO-15 atoms-DBCO, DBCO-26 atoms-DBCO, DBCO-35 atoms-DBCO, DBCO-PEG4-S-S-PEG3-biotin, DBCO-S-S-PEG3-biotin, DBCO-S-S-PEG11-biotin and (succinimidyl 3-(2-pyridyldithio)propionate (SPDP).

In certain embodiments, a covalent linkage refers to the linkage between two or more amino acids. One or more of the linked amino acids may be naturally occurring or non-naturally occurring. One or more of the linked amino acids may be chemically modified.

As used herein, a “natural amino acid” refers to the twenty genetically encoded alpha-amino acids. See, e.g., Biochemistry by L. Stryer, 3^(rd) ed. 1988, Freeman and Company, New York for structures of the twenty natural amino acids.

As used herein, an “unnatural amino acid,” “modified amino acid” or “chemically modified amino acid” refers to any amino acid, modified amino acid, or amino acid analogue other than the twenty genetically encoded alpha-amino acids. Unnatural amino acids have side chain groups that distinguish them from the natural amino acids, although unnatural amino acids can be naturally occurring compounds other than the twenty proteinogenic alpha-amino acids. In addition to side chain groups that distinguish them from the natural amino acids, unnatural amino acids may have an extended backbone such as beta-amino acids.

Non-limiting examples of unnatural amino acids include selenocysteine, pyrrolysine, homocysteine, an O-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, an O-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, an isopropyl-L-phenylalanine, a p-azido-L-phenylalanine, a p-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine, a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, a p-bromophenylalanine, a p-amino-L-phenylalanine, an isopropyl-L-phenylalanine, an unnatural analogue of a tyrosine amino acid; an unnatural analogue of a glutamine amino acid; an unnatural analogue of a phenylalanine amino acid; an unnatural analogue of a serine amino acid; an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analogue containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar-containing amino acid; a redox-active amino acid; an α-hydroxy containing acid; an amino thio acid containing amino acid; an α,α disubstituted amino acid; a β-amino acid; and a cyclic amino acid other than proline. In an embodiment of the helicases described herein, one or more amino acids of the helicase are substituted with one or more unnatural amino acids and/or one or more natural amino acids.

In certain embodiments, a helicase-_(X) is a closed form, conformationally-constrained helicase monomer generated from a helicase polypeptide that was reacted with an intramolecular crosslinking agent. In certain embodiments, a helicase-_(Y) is an open form, conformationally-constrained helicase monomer generated from a helicase polypeptide that was reacted with an intramolecular crosslinking agent.

The chemical structures described herein are named according to IUPAC nomenclature rules and include art-accepted common names and abbreviations where appropriate. The IUPAC nomenclature can be derived with chemical structure drawing software programs, such as ChemDraw® (PerkinElmer, Inc.), ChemDoodle® (iChemLabs, LLC) and Marvin (ChemAxon Ltd.). The chemical structure controls in the disclosure to the extent that an IUPAC name is misnamed or otherwise conflicts with the chemical structure disclosed herein. E. coli Rep mutants can be engineered that are intramolecularly crosslinked to constrain the 2B subdomain in open or closed conformations. Residues for the cysteine substitution mutagenesis and the length of the bis-maleimide crosslinkers were selected such that when crosslinked, the 2B subdomain cannot rotate appreciably, effectively locking the protein in one conformation (FIG. 1A, B). The closed fix in of a helicase that is crosslinked in a constrained conformation is denoted with the suffix “−X”, and the open form of a helicase that is crosslinked in a constrained conformation is denoted with the suffix “−Y.” For Rep, Rep-X and Rep-Y represent the conformationally-constrained closed and open forms, respectively. Enzymatic activities of Rep-X and Rep-Y monomers were studied in single molecule and ensemble assays employing fluorescence resonance energy transfer (FRET), total internal reflection fluorescence (TIRF) microscopy, and optical tweezers force spectroscopy.

The Rep mutant sequences used to generate Rep-X and Rep-Y include those nucleotide and amino acid sequences identified in Table 1.

TABLE 1  Amino Acid and Nucleotide Sequences for exemplary Rep-X and Rep-Y proteins Polypeptide/DNA/RNA 5′→3′ (nucleotide sequence)  (SEQ ID NO:_) N→C (amino acid sequence) Wild type Rep helicase ATGCGTCTAAACCCCGGCCAACAACAAGCTGTCGAATTCGTT (gene sequence) ACCGGCCCCTGCCTGGTGCTGGCGGGCGCGGGTTCCGGTAAA >gi|556503834:3960677- ACTCGTGTTATCACCAATAAAATCGCCCATCTGATCCGCGGTT 3962698 GCGGTTATCAGGCGCGGCACATTGCGGCGGTGACCTTTACTA Escherichia coli ATAAAGCAGCGCGCGAGATGAAAGAGCGTGTAGGGCAGACG str. K-12 CTGGGGCGCAAAGAGGCGCGTGGGCTGATGATCTCCACTTTC substr. MG1655, CATACGTTGGGGCTGGATATCATCAAACGCGAGTATGCGGCG complete genome CTTGGGATGAAAGCGAACTTCTCGTTGTTTGACGATACCGATC (SEQ ID NO: 31) AGCTTGCTTTGCTTAAAGAGTTGACCGAGGGGCTGATTGAAG ATGACAAAGTTCTCCTGCAACAACTGATTTCGACCATCTCTAA CTGGAAGAATGATCTCAAAACACCGTCCCAGGCGGCAGCAAG TGCGATTGGCGAGCGGGACCGTATTTTTGCCCATTGTTATGGG CTGTATGATGCACACCTGAAAGCCTGTAACGTTCTCGACTTCG ATGATCTGATTTTATTGCCGACGTTGCTGCTGCAACGCAATGA AGAAGTCCGCAAGCGCTGGCAGAACAAAATTCGCTATCTGCT GGTGGATGAGTATCAGGACACCAACACCAGCCAGTATGAGCT GGTGAAACTGCTGGTGGGCAGCCGCGCGCGCTTTACCGTGGT GGGTGACGATGACCAGTCGATCTACTCCTGGCGCGGTGCACG TCCGCAAAACCTGGTGCTGCTGAGTCAGGATTTTCCGGCGCTG AAGGTGATTAAGCTTGAGCAGAACTATCGCTCTTCCGGGCGT ATTCTGAAAGCGGCGAACATCCTGATCGCCAATAACCCGCAC GTCTTTGAAAAGCGTCTGTTCTCCGAACTGGGTTATGGCGCGG AGCTAAAAGTATTAAGCGCGAATAACGAAGAACATGAGGCTG AGCGCGTTACTGGCGAGCTGATCGCCCATCACTTCGTCAATAA AACGCAGTACAAAGATTACGCCATTCTTTATCGCGGTAACCAT CAGTCGCGGGTGETTGAAAAATTCCTGATGCAAAACCGCATC CCGTACAAAATATCTGGTGGTACGTCGTTTTTCTCTCGTCCTG AAATCAAGCACTTGCTGGCTTATCTGCGCGTGCTGACTAACCC GGACGATGACAGCGCATTTCTGCGTATCGTTAACACGCCGAA GCGAGAGATTGGCCCGGCTACGCTGAAAAAGCTGGGTGAGTG GGCGATGACGCGCAATAAAAGCATGTTTACCGCCAGCTTTGA TATGGGCCTGAGTCAGACGCTTAGCGGACGTGGTTATGAAGC ATTGACCCGCTTCACTCACTGGTTGGCAGAAATCCAGCGTCTG GCGGAGCGGGAGCCGATTGCCGCGGTGCGTGATCTGATCCAT GGCATGGATTATGAATCCTGGCTGTACGAAACATCGCCCAGC CCGAAAGCCGCCGAAATGCGCATGAAGAACGTCAACCAACTG TTTAGCTGGATGACGGAGATGCTGGAAGGCAGTGAACTGGAT GAGCCGATGACGCTCACCCAGGTGGTGACGCGCTTTACTTTGC GCGACATGATGGAGCGTGGTGAGAGTGAAGAAGAGCTGGATC AGGTGCAACTGATGACTCTCCACGCGTCGAAAGGGCTGGAGT TTCCTTATGTCTACATGGTCGGTATGGAAGAAGGGTTTTTGCC GCACCAGAGCAGCATCGATGAAGATAATATCGATGAGGAGCG GCGGCTGGCCTATGTCGGCATTACCCGCGCCCAGAAGGAATT GACCTTTACGCCTGTGTAAAGAACGCCGTCAGTACGGCGAACT GGTGCGCCCGGAGCCGAGCCGCTTTTTGCTGGAGCTGCCGCA GGATGATCTGATTTGGGAACAGGAGCGCAAAGTGGTCAGCGC CGAAGAACGGATGCAGAAAGGGCAAAGCCATCTGGCGAATCT GAAAGCGATGATGGCGGCAAAACGAGGGAAATAA Wild type Rep helicase MRLNPGQQQAVEFVTGPCLVLAGAGSGKTRVITNKIAHLIRGCG (amino acid sequence) YQARHIAAVTFTNKAAREMKERVGQTLGRKEARGLMISTFHTLG >gi|48994965|gb  LDIIKREYAALGMKANFSLFDDTDQLALLKELTEGLIEDDKVLLQ AAT48209.1|DNA helicase  QLISTISNWKNDLKTPSQAAASAIGERDRIFAHCYGLYDAHLKAC and single-stranded  NVLDFDDLILLPTLLLQRNEEVRKRWQNKIRYLLVDEYQDTNTS DNA-dependent ATPase QYELVKLLVGSRARFTVVGDDDQSIYSWRGARPQNLVLLSQDFP [Escherichia coli str.  ALKVIKLEQNYRSSGRILKAANILLANNPHVFEKRLFSELGYGAEL K-12 substr. MG16] KVLSANNEEHEAERVTGELIAHHFVNKTQYKDYAILYRGNHQSR (SEQ ID NO: 32) VFEKFLMQNRIPYKISGGTSFFSRPEIKDLLAYLRVLTNPDDDSAF LRIVNTPKREIGPATLKKLGEWAMTRNKSMFTASFDMGLSQTLS GRGYEALTRFTHWLAEIQRLAEREPIAAVRDLIHGMDYESWLYE TSPSPKAAEMRMKNVNQLFSWMTEMLEGSELDEPMTLTQVVTR FTLRDMMERGESEEELDQVQLMTLHASKGLEFPYVYMVGMEEG FLPHQSSIDEDNIDEERRLAYVGITRAQKELTFTLCKERRQYGELV RPEPSRFLLELPQDDLIWEQERKVVSAEERMQKGQSHLANLKAM MAAKRGK Rep-_(X) polypmtide¹ MRLNPGQQQAVEFVTGPLLVLAGAGSGKTRVITNKIAHLIRGSG (SEQ ID NO: 1) YQARHIAAVTFTNKAAREMKERVGQTLGRKEARGLMISTFHTLG LDIIKREYAALGMKANFSLFDDTDQLALLKELTEGLIEDDKVLLQ QLISTISNWKNDLKTPSQAAASAIGERDRIFAHVYGLYDAHLKAC NVLDFDDLILLPTLLLQRNEEVRKRWQNKIRYLLVDEYQDTNTS QYELVKLLVGSRARFTWGDDDQSIYSWRGARPQNLVLLSQDFP ALKVIKLEQNYRSSGRILKAANILIANNPHVFEKRLFSELGYGAEL KVLSANNEEHEAERVTGELLAHHFVNKTQYKDYAILYRGNHQSR VFEKFLMQNRIPYKISGGTSFFSRPEIKDLLAYLRVLTNPDDDCAF LRIVNTPKREIGPATLKKLGEWAMTRNKSMFTASFDMGLSQTLS GRGYEALTRFTHWLAEIQRLAEREPIAAVRDLIHGMDYESWLYE TSPSPKAAEMRMKNVNQLFSWNTEMLEGSELDEPMTLTQVVTR FTLRDMMERGESEEELDQVQLMTLHASKGLEFPYVYMVGMEEG FLPHQSSIDEDNIDEERRLAYVGITRAQKELTFTLAKERRQYGELV RPEPSRFLLELPQDDLIWEQERKVVSAEERMQKGQSHLANLKAM MAAKRGK Rep-_(X) DNA² ATGCGTCTAAACCCCGGCCAACAACAAGCTGTCGAATTCGTT (SEQ ID NO: 2) ACCGGCCCCTTGCTGGTGCTGGCGGGCGCGGGTTCCGGTAAA ACTCGTGTTATCACCAATAAAATCGCCCATCTGATCCGCGGTA GCGGGTACCAGGCGCGGCACATTGCGGCGGTGACCTTTACTA ATAAAGCAGCGCGCGAGATGAAAGAGCGTGTAGGGCAGACG CTGGGGCGCAAAGAGGCGCGTGGGCTGATGATCTCCACTTTC CATACGTTGGGGCTGGATATCATCAAACGCGAGTATGCGGCG CTTGGGATGAAAGCGAACTTCTCGTTGTTTGACGATACCGATC AGCTTGCTTTGCTTAAAGAGTTGACCGAGGGGCTGATTGAAG ATGACAAAGTTCTCCTGCAACAACTGATTTCGACCATCTCTAA CTGGAAGAATGATCTCAAAACACCGTCCCAGGCGGCAGCAAG TGCGATTGGCGAGCGGGACCGTATTTTTGCCCATGTTTATGGG CTGTATGATGCACACCTGAAAGCCTGTAACGTTCTCGACTTCG ATGATCTGATTTTATTGCCGACGTTGCTGCTGCAACGCAATGA AGAAGTCCGCAAGCGCTGGCAGAACAAAATTCGCTATCTGCT GGTGGATGAGTATCAGGACACCAACACCAGCCAGTATGAGCT GGTGAAACTGCTGGTGGGCAGCCGCGCGCGCTTTACCGTGGT GGGTGACGATGACCAGTCGATCTACTCCTGGCGCGGTGCACG TCCGCAAAACCTGGTGCTGCTGAGTCAGGATTTTCCGGCGCTG AAGGTGATTAAGCTTGAGCAGAACTATCGCTCTTCCGGGCGT ATTCTCAAAGCGGCGAACATCCTGATCGCCAATAACCCGCAC GTCTTTGAAAAGCGTCTGTTCTCCGAACTGGGTTATGGCGCGG ACTCTAAAAGTATTAAGCGCGAATAACGAAGAACATGAGGCTG AGCGCGTTACTGGCGAGCTGATCGCCCATCACTTCGTCAATAA AACGCAGTACAAAGATTACGCCATTCTTTATCGCGGTAACCAT CAGTCGCGGGTGTTTGAAAAATTCCTGATGCAAAACCGCATC CCGTACAAAATATCTGGTGGTACGTCGTTTTTCTCTCGTCCTG AAATCAAGGACTTGCTGGCTTATCTGCGCGTGCTGACTAACCC GGACGATGACTGCGCATTTCTGCGTATCGTTAACACGCCGAA GCGAGAGATTGGCCCGGCTACGCTGAAAAAGCTGGGTGAGTG GGCGATGACGCGCAATAAAAGCATGTTTACCGCCAGCTTTGA TATGGGCCTGAGTCAGACGCTTAGCGGACGTGGTTATGAAGC ATTGACCCGCTTCACTCACTGGTTGGCAGAAATCCAGCGTCTG GCGGAGCGGGAGCCGATTGCCGCGGTGCGTGATCTGATCCAT GGCATGGATTATGAATCCTGGCTGTACGAAACATCGCCCAGC CCGAAAGCCGCCGAAATGCGCATGAAGAACGTCAACCAACTG TTTAGCTGGATGACGGAGATGCTGGAAGGCAGTGAACTGGAT GAGCCGATGACGCTCACCCAGGTGGTGACGCGCTTTACTTTGC GCGACATGATGGAGCGTGGTGAGAGTGAAGAAGAGCTGGATC AGGTGCAACTGATGACTCTCCACGCGTCGAAAGGGCTGGAGT TTCCTTATGTCTACATGGTCGGTATGGAAGAAGGGTTTTTGCC GCACCAGAGCAGCATCGATGAAGATAATATCGATGAGGAGCG GCGGCTGGCCTATGTCGGCATTACCCGCGCCCAGAAGGAATT GACCTTTACGCTGGCTAAAGAACGCCGTCAGTACGGCGAACT GGTGCGCCCGGAGCCGAGCCGCTTTTTGCTGGAGCTGCCGCA GGATGATCTGATTTGGGAACAGGAGCGCAAAGTGGTCAGCGC CGAAGAACGGATGCAGAAAGGGCAAAGCCATCTGGCGAATCT GAAAGCGATGATGGCGGCAAAACGAGGGAAATAA Rep-_(X) RNA³ AUGCGUCUAAACCCCGGCCAACAACAAGCUGUCGANUUCGU (SEQ ID NO: 3) UACCGGCCCCUUGCUGGUGCUGGCGGGCGCGGGUUCCGGUA AAACUCGUGUUAUCACCAAUAAAAUCGCCCAUCUGAUCCGC GGUAGCGGGUACCAGGCGCGGCACAUUGCGGCGGUGACCUU UACUAAUAAAGCAGCGCGCGAGAUGAAAGAGCGUGUAGGGC AGACGCUGGGGCGCAAAGAGGCGCGUGGGCUGAUGAUCUCC ACUUUCCAUACGUUGGGGCUGGAUAUCAUCAAACGCGAGUA UGCGGCGCUUGGGAUGAAAGCGAACUUCUCGUUGUUUGACG AUACCGAUCAGCUUGCUUUGCUUAAAGAGUUGACCGAGGGG CUGAUUGAAGAUGACAAAGUUCUCCUGCAACAACUGAUUUC GACCAUCUCUAACUGGAAGAAUGAUCUCAAAACACCGUCCC AGGCGGCAGCAAGUGCGAUUGQCGAGCGGGACCGUAUUUUU GCCCAUGUUUAUGGGCUGUAUGAUGCACACCUGAAAGCCUG UAACGUUCUCGACUUCGAUGAUCUGAUUUUAUUGCCGACGU UGCUGCUGCAACGCAAUGAAGAAGUCCGCAAGCGCUGGCAG AACAAAAUUCGCUAUCUGCUGGUGGAUGAGUAUCAGGACAC CAACACCAGCCAGUAUGAGCUGGUGAAACUGCUGGUGGGCA GCCGCGCGCGCUUUACCGUGGUGGGUGACGAUGACCAGUCG AUCUACUCCUGGCGCGGUGCACGUCCGCAAAACCUGGUGCU GCUGAGUCAGGAUUUUCCGGCGCUGAAGGUGAUUAAGCUUG AGCAGAACUAUCGCUCUUCCGUGCGUAUUCUGAAAGCGGCG AACAUCCUGAUCGCCAAUAACCCGCACGUCUUUGAAAAGCG UCUGUUCUCCGAACUGGGUUAUGGCGCGGAGCUAAAAGUAU UAAGCGCGAAUAACGAAGAACAUGAGGCUGAGCGCGUUACU GGCGAGCUGAUCGCCCAUCACUUCGUCAAUAAAACGCAGUA CAAAGNUUACGCCAUUCUUUAUCGCGGUAACCAUCAGUCGC GGGUGUUUGAAAAAUUCCUGAUGCAAAACCGCAUCCCGUAC AAAAUAUCUGGUGGUACGUCGUUUUUCUCUCGUCCUGAAAU CAAGGACUUGCUGGCUUAUCUGCGCGUGCUGACUAACCCGG ACGAUGACUGCGCAUUUCUGCGUAUCGUUAACACGCCGAAG CGAGAGAUUGGCCCGGCUACGCUGAAAAAGCUGGGUGAGUG GGCCAUGACGCGCAAUAAAAGCAUGUUUACCGCCAGCUUUG AUAUGGGCCUGAGUCAGACGCUUAGCGGACGUGGUUAUGAA GCAUUGACCCGCUUCACUCACUGGUUGGCAGAAAUCCAGCG UCUGGCGGAGCGGGAGCCGAUUGCCGCGGUGCGUGAUCUGA UCCAUGGCAUGGAUUAUGAAUCCUGGCUGUACGAAACAUCG CCCAGCCCGAAAGCCGCCGAAAUGCGCAUGAAGAACGUCAA CCAACUGUULAGCUGGAUGACGGAGAUGCUGGAAGGCAGUG AACUGGAUGAGCCGAUGACGCUCACCCAGGUGGUGACGCGC UUUACUUUGCGCGACAUGAUGGAGCGUGGUGAGAGUGAAG AAGAGCUGGAUCAGGUGCAACUGAUGACUCUCCACGCGUCG AAAGGGCUGGAGUUUCCUUAUGUCUACAUGGUCGGUAUGG AAGAAGGGUUUUUGCCGCACCAGAGCAGCAUCGAUGAAGAU AAUAUCGAUGAGGAGCGGCGGCUGGCCUAUGUCGGCAUUAC CCGCGCCCAGAAGGAAUUGACCUUUACGCUGGCUAAAGAAC GCCGUCAGUACGGCGAACUGGUGCGCCCGGAGCCGAGCCGC UUUUUGCUGGAGCUGCCGCAGGAUGAUCUGAUUUGUGAACA GGAGCGCAAAGUGGUCAGCGCCGAAGAACGGAUGCAGAAAG GGCAAAGCCAUCUGGCGAAUCUGAAAGCGAUGAUGGCGGCA AAACGAGGGAAAUAA Rep-_(X) polypeptide⁴ SEQ ID NO: 1 and formula no 2 in Table 2  (SEQ ID NO: 4) (1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2, 5-dione) Rep-_(Y) polypeptide⁵ MRLNPGQQQAVEFVTGPLLVLAGAGSGKTRVITNKIAHLIRGSG (SEQ ID NO: 5) YQARHIAAVTFTNKAAREMKERVGQTLGRKEARGLMISTFHTLG LDIIKREYAALGMKANFSLFDDTDQLALLKELTEGLIEDDKVLLQ QLISTISNWKNDLKTPSQAAASAIGERDRIFAHVYGLYDAHLKAC NVLDFDDLILLPTLLLQRNEEVRKRWQNKIRYLLNDEYQDTNTS QYELVKLLVGSRARFTVVGDDDQSIYSWRGARPQNLVLLSQDFP ALKVIKLEQNYRSSGRILKAANILIANNPHVFEKRLFSELGYGAEL KVLSANNEEHEAERVTGELIAHHFVNKTQYKDYAILYRGNHQSR VFEKFLMQNRIPYKISGGTSFFSRPEIKDLLAYLRVLTNPDDDCAF LRIVNTPKREIGPATLKKLGEWAMTRNKSMFTASFDMGLSQTLS GRGYEALTRFTHWLAEIQRLAEREPIAAVRDLIHGMDYESWLYE TSPSPKAAEMRMKNVNQLFSWMTEMLEGSELDEPMTLTQVVTR FTLRDMMERGESEEELDQVQLMTLHASKGLEFPYVYMVGMEEG FLPHQSSIDEDNIDEERRLAYVGITRAQKELTFTLAKERRQYGELV RPEPSRFLLELPDDLIWEQERKVVSAEERMQKGQSHLANLKAM MAAKRGK Rep-_(Y) DNA⁶ ATGCGTCTAAACCCCGGCCAACAACAAGCTGTCGAATTCGTT (SEQ ID N0:6) ACCGGCCCCTTGCTGGTGCTGGCGGGCGCGGGTTCCGGTAAA ACTCGTGTTATCACCAATAAAATCGCCCATCTGATCCGCGGTA GCGGGTACCAGGCGCGGCACATTGCGGCGGTGACCTTTACTA ATAAAGCAGCGCGCGAGATGAAAGAGCGTGTAGGGCAGACG CTGGGGCGCAAAGAGGCGCGTGGGCTGATGATCTCCACTTTC CATACGTTGGGGCTGGATATCATCAAACGCGAGTATGCGGCG CTTGGGATGAAAGCGAACTTCTCGTTGTTTGACGATACCGATC AGCTTGCTTTGCTTAAAGAGTTGACCGAGGGGCTGATTGAAG ATGACAAAGTTCTCCTGCAACAACTGATTTCGACCATCTCTAA CTGGAAGAATGATCTCAAAACACCGTCCCAGGCGGCAGCAAG TGCGATTGGCGAGCGGGACCGTATTTTTGCCCATGTTTATGGG CTGTATGATGCACACCTGAAAGCCTGTAACGTTCTCGACTTCG ATGATCTGATTTTATTGCCGACGTTGCTGCTGCAACGCAATGA AGAAGTCCGCAAGCGCTGGCAGAACAAAATTCGCTATCTGCT GGTGGATGAGTATCAGGACACCAACACCAGCCAGTATGAGCT GGTGAAACTGCTGGTGGGCAGCCGCGCGCGCTTTACCGTGGT GGGTGACGATGACCAGTCGATCTACTCCTGGCGCGGTGCACG TCCGCAAAACCTGGTGCTGCTGAGTCAGGATTTTCCGGCGCTG AAGGTGATTAAGCTTGAGCAGAACTATCGCTCTTCCGGGCGT ATTCTGAAAGCGGCGAACATCCTGATCGCCAATAACCCGCAC GTCTTTGAAAAGCGTCTGTTCTCCGAACTGGGTTATGGCGCGG AGCTAAAAGTATTAAGCGCGAATAACGAAGAACATGAGGCTG AGCGCGTTACTGGCGAGCTGATCGCCCATCACTTCGTCAATAA AACGCAGTACAAAGATTACGCCATTCTTTATCGCGGTAACCAT CAGTCGCGGGTGTTTGAAAAATTCCTGATGCAAAACCGCATC CCGTACAAAATATCTGGTGGTACGTCGTTTTTCTCTCGTCCTG AAATCAAGGACTTGCTGGCTTATCTGCGCGTGCTGACTAACCC GGACGATGACTGCGCATTTCTGCGTATCGTTAACACGCCGAA GCGAGAGATTGGCCCGGCTACGCTGAAAAAGCTGGGTGAGTG GGCGATGACGCGCAATAAAAGCATGTTTACCGCCAGCTTTGA TATGGGCCTGAGTCAGACGCTTAGCGGACGTGGTTATGAAGC ATTGACCCGCTTCACTCACTGGTTGGCAGAAATCCAGCGTCTG GCGGAGCGGGAGCCGATTGCCGCGGTGCGTGATCTGATCCAT GGCATGGATTATGAATCCTGGCTGTACGAAACATCGCCCAGC CCGAAAGCCGCCGAAATGCGCATGAAGAACGTCAACCAACTG TTTAGCTGGATGACGGAGATGCTGGAAGGCAGTGAACTGGAT GAGCCGATGACGCTCACCCAGGTGGTGACGCGCTTTACTTTGC GCGACATGATGGAGCGTGGTGAGAGTGAAGAAGAGCTGGATC AGGTGCAACTGATGACTCTCCACGCGTCGAAAGGGCTGGAGT TTCCTTATGTCTACATGGTCGGTATGGAAGAAGGGTTTTTGCC GCACCAGAGCAGCATCGATGAAGATAATATCGATGAGGAGCG GCGGCTGGCCTATGTCGGCATTACCCGCGCCCAGAAGGAATT GACCTTTACGCTGGCTAAAGAACGCCGTCAGTACGGCGAACT GGTGCGCCCGGAGCCGAGCCGCTTTTTGCTGGAGCTGCCGCA GGATGATCTGATTTGGGAACAGGAGCGCAAAGTGGTCAGCGC CGAAGAACGGATGCAGAAAGGGCAAAGCCATCTGGCGAATCT GAAAGCGATGATGGCGGCAAAACGAGGGAAATAA Rep-_(Y) RNA⁷ AUGCGUCUAAACCCCGGCCAACAACAAGCUGUCGAAUUCGU (SEQ ID NO: 7) UACCGGCCCCUUGCUGGUGCUGGCGGGCGCGGGUUCCGGUA AAACUCGUGUUAUCACCAAUAAAAUCGCCCAUCUGAUCCGC GGUAGCGGGUACCAGGCGCGGCACAUUGCGGCGGUGACCUU UACUAAUAAAGCAGCGCGCGAGAUGAAAGAGCGUGUAGGGC AGACGCUGGGGCGCAAAGAGGCGCGUGGGCUGAUGAUCUCC ACUUUCCAUACGUUGGGGCUGGAUAUCAUCAAACGCGAGUA UGCGGCGCUUGGGAUGAAAGCGAACUUCUCGUUGUUUGACG AUACCGAUCAGCUUGCUUUGCUUAAAGAGUUGACCGAGGGG CUGAUUGAAGAUGACAAAGUUCUCCUGCAACAACUGAUUUC GACCAUCUCUAACUGGAAGAAUGAUCUCAAAACACCGUCCC AGGCGGCAGCAAGUGCGAUUGGCGAGCGGGACCGUAUUUUU GCCCAUGUUUAUGGGCUGUAUGAUGCACACCUGAAAGCCUG UAACGUUCUCGACUUCGAUGAUCUGNUUUUAUUGCCGACGU UGCUGCUGCAACGCAAUGAAGAAGUCCGCAAGCGCUGGCAG AACAAAAUUCGCUAUCUGCUGGUGGAUGAGUAUCAGGACAC CAACACCAGCCAGUAUGAGCUGGUGAAACUGCUGGUGGGCA GCCGCGCGCGCUUUACCGUGGUGGGUGACGAUGACCAGUCG AUCUACUCCUGGCGCGGUGCACGUCCGCAAAACCUGGUGCU GCUGAGUCAGGAUUUUCCGGCGCUGAAGGUGAUUAAGCUUG AGCAGAACUAUCGCUCUUCCGGGCGUAUUCUGAAAGCGGCG AACAUCCUGAUCGCCAAUAACCCGCACGUCUUUGAAAAGCG UCUGUUCUCCGAACUGGGUUAUGGCGCGGAGCUAAAAGUAU UAAGCGCGAAUAACGAAGAACAUGAGGCUGAGCGCGUUACU GGCGAGCUGAUCGCCCAUCACUUCGUCAAUAAAACGCAGUA CAAAGAUUACGCCAUUCUUUAUCGCGGUAACCAUCAGUCGC GGGUGUUUGAAAAAUUCCUGAUGCAAAACCGCAUCCCGUAC AAAAUAUCUGGUGGUACGUCGUUUUUCUCUCGUCCUGAAAU CAAGGACUUGCUGGCUUAUCUGCGCGUGCUGACUAACCCGG ACGAUGACUGCGCAUUUCUGCGUAUCGUUAACACGCCGAAG CGAGAGAUUGGCCCGGCUACGCUGAAAAAGCUGGGUGAGUG GGCGAUGACGCGCAAUAAAAGCAUGUUUACCGCCAGCUUUG AUAUGGGCCUGAGUCAGACGCUUAGCGGACGUGGUUAUGAA GCAUUGACCCGCUUCACUCACUGGUUGGCAGAAAUCCAGCG UCUGGCGCAGCGGCAGCCGAUUGCCGCGGUGCGUGAUCUGA UCCAUGGCAUGGAUUAUGAAUCCUGGCUGUACGAAACAUCG CCCAGCCCGAAAGCCGCCGAAAUGCGCAUGAAGAACGUCAA CCAACUGUUUAGCUGGAUGACGGAGAUGCUGGAAGGCAGUG AACUGGAUGAGCCGAUGACGCUCACCCAGGUGGUGACGCGC UUUACUUUGCGCGACAUGAUGGAGCGUGGUGAGAGUGAAG AAGAGCUGGAUCAGGUGCAACUGAUGACUCUCCACGCGUCG AAAGGGCUGGAGUUUCCUUAUGUCUACAUGGUCGGUAUGG AAGAAGGGUUUUUGCCGCACCAGAGCAGCAUCGAUGAAGAU AAUAUCCAUGAGGAGCGGCGGCUGGCCUAUGUCGGCAUUAC CCGCGCCCAGAAGGAAUUGACCUUUACGCUGGCUAAAGAAC GCCGUCAGUACGGCGAACUGGUGCGCCCGGAGCCGAGCCGC UUUUUGCUGGAGCUGCCGCAGGAUGAUCUGAUUUGGGAACA GGAGCGCAAAGUGGUCAGCGCCGAAGAACGGAUGCAGAAAG GGCAAAGCCAUCUGGCGAAUCUGAAAGCGAUGAUGGCGGCA AAACGAGGGAAAUAA Rep-_(Y) polypeptide⁸ SEQ ID NO: 5 and formula no 2 in Table 2  (SEQ ID NO: 8) (1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2, 5-dione). ¹This Rep mutant encodes mutations removing natural cysteine residues found in the wild-type Rep and include further amino acid mutations to facilitate intramolecular crosslinking to an intramolecular crosslinking agent to generate the Rep-x polypeptide. ²The DNA sequence corresponds to the open reading frame that encodes the polypeptide of SEQ ID NO: 1. ³The RNA sequence corresponds to the open reading frame that encodes the polypeptide of SEQ ID NO: l. ⁴The Rep-_(X) polypeptide closed foim monomer following reaction of Repx polypeptide (SEQ ID NO: 1) with an intramolecular crosslinking agent: ⁵This Rep mutant encodes mutations that remove natural cysteine residues found in the wild-type Rep and include further amino acid mutations to facilitate intramolecular crosslinking to an intramolecular crosslinking agent to generate the Rep-y polypeptide. ⁶The DNA sequence corresponds to the open reading frame that encodes the polypeptide of SEQ ID NO: 5. ⁷The RNA sequence corresponds to the open reading frame that encodes the polypeptide of SEQ ID NO: 5. ⁸The Rep-_(Y) polypeptide open forui monomer following reaction of Repy polypeptide (SEQ ID NO: 5) with an intramolecular crosslinking agent:

The intramolecular crosslinking agents suitable for generating versions of Rep-_(X) and Rep-_(Y) include those identified in Table 2.

TABLE 2 Exemplary intramolecular crosslinking agents for generating Rep-_(X) and Rep-_(Y) Formula No. Compound Structure (IUPAC Name) 1

2

3

4

5

6

These intramolecular crosslinking agents yield intramolecular crosslinked monomer structures when reacted with Rep-_(X) and Rep-_(Y) polypeptides. The linkers can have a length in the range from about 6 Å to about 25 Å. These types of linkers have an alkyl length in the range corresponding from about C₇ to about C₂₀, wherein highly preferred linkers have a length in the range from about C₁₀ to about C₁₂. Methods and conditions for generating intramolecular crosslink formation in proteins are well known in the art for these types of intramolecular crosslinking agents, and such methods and conditions are applicable to the helicases of this disclosure.

Rep-_(X) would be inefficient in DNA unwinding even at high concentrations that make the wild type Rep active if the closed for in is inactive for unwinding. In multiple turnover ensemble unwinding reactions using FRET-labeled DNA (see, for example, FIG. 1C), however, Rep-X unwound an 18-bp substrate with a 3′-(dT)₁₀ overhang (SEQ ID NO: 33) at a much faster rate and higher reaction amplitude than the wild type Rep (FIG. 1D). In contrast, Rep-Y unwinding rates were similar to that of Rep (FIG. 1E), indicating that the dramatic unwinding enhancement is specifically achieved in the closed conformation. Because the large enhancement in unwinding activity observed in bulk solution can result from the activation of a monomer or from enhanced oligomerization, single molecule FRET (smFRET) experiments were performed to test if a single Rep-X can unwind DNA.

Rep and Rep-X monomers were immobilized to a polymer-passivated quartz surface using antibodies against the N-terminal hexa-histidine-tag (SEQ ID NO: 36) on the protein (FIG. 2A) to ensure that the observed activity belonged to monomers (T. Ha et al., Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638-641 (2002)). For the unwinding substrate, we used a 18-bp duplex DNA with a 3′-(dT)₂₀ overhang (SEQ ID NO: 37) labeled with a donor (Cy3) and an acceptor (Cy5) at two opposite ends of the DNA duplex, allowing us to identify unwinding reactions as increases in FRET efficiency (E_(FRET)) (FIG. 2A) (G. Lee, M. A. Bratkowski, F. Ding A. Ke, T. Ha, Elastic Coupling Between RNA Degradation and Unwinding by an Exoribonuclease. Science (New York, N.Y. 336, 1726-1729 (2012)). When the DNA and ATP were added to the reaction chamber, we could observe the capture of a single DNA molecule by a single protein as the sudden appearance of fluorescence signal (FIG. 2B-E). Subsequent DNA unwinding generated ssDNA strands that coil up due to high flexibility and E_(FRET) increased (M. C. Murphy, I. Rasnik, W. Cheng, T. M. Lohman, T. Ha, Probing single-stranded DNA conformational flexibility using fluorescence spectroscopy. Biophysical journal 86, 2530-2537 (2004)). Once the duplex was completely unwound, the acceptor-labeled strand was released, which was marked by sudden disappearance of the acceptor signal and recovery of the donor signal. The donor-labeled strand then dissociated, resulting in complete loss of fluorescence. The mean duration of unwinding measured from the E_(FRET) increase to acceptor strand release was ˜0.6 s, giving a lower limit on the unwinding speed of 30 bp/s for the 18-bp substrate (FIG. 2F). About 82% of the DNA molecules (661 out of 809) that initially bound to Rep-X monomers were unwound (FIG. 2G). In contrast, only 2% of the DNA molecules (13 out of 847) that bound to Rep (i.e. without crosslinking) showed unwinding, and the unwinding yield for Rep-Y was 16% (357 out of 2212) (FIG. 2G), showing that constraining Rep into the closed form selectively activates the unwinding activity of a monomer. The nonzero amplitude of unwinding for Rep and Rep-Y may be due to conformational constraints caused by surface tethering in a small fraction of molecules.

In vitro studies have shown that the unwinding processivity of Rep and related helicases is limited even in their oligomeric forms, ranging from 30-50 bp (A. Niedziela-Majka, M. A. Chesnik, E. J. Tomko, T. M. Lohman, Bacillus stearothermophilus PcrA monomer is a single-stranded DNA translocase but not a processive helicase in vitro. The Journal of biological chemistry 282, 27076-27085 (2007); Ha et al (2008) supra; J. A. Ali, T. M. Lohman, Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase. Science (New York, N.Y. 275, 377-380 (1997)). In order to investigate the processivity of Rep-X, we employed a dual optical tweezers assay (FIG. 3A; J. R. Moffitt et al., Intersubunit coordination in a homomeric ring ATPase. Nature 457, 446-450 (2009)) that can monitor unwinding amplitudes and speeds over thousands of base pairs of DNA. The two traps held two streptavidin functionalized sub-micron sized polystyrene beads. The first was coated with 6-kbp dsDNA attached via a biotin on the blunt end and containing a 3′ poly-dT ssDNA overhang on the other end ((dT)₁₀ (SEQ ID NO: 33), (dT)₁₅ (SEQ ID NO: 34), and (dT)₇₅ (SEQ ID NO: 35) see Example 7)). The other bead was coated with Rep-X molecules via biotinylated antibody against the hexa-histidine-tag (SEQ ID NO: 36). A laminar flow cell with two parallel streams of buffer was created for controlling the initiation of the unwinding reaction (inset of FIG. 3B; L. R. Brewer, P. R. Bianco, Laminar flow cells for single-molecule studies of DNA-protein interactions. Nature methods 5, 517-525 (2008)). When the two beads were brought in proximity in the first laminar stream (Buffer C with 100 μM ATP and 100 μM ATP-γS), a single Rep-X binding to the 3′ overhang of the DNA formed a tether between the two beads without initiating unwinding. When the tethered beads were moved to the second laminar stream (Buffer C and 1 mM ATP), the DNA tether between the beads progressively shortened as the Rep-X monomer unwound and pulled the DNA. Unless otherwise stated, SSB was added to the second laminar stream in order to prevent any subsequent interaction of unwound ssDNA with other Rep-X on the bead surface. The optical tweezers experiments that were performed without SSB yielded the same Rep-X behavior (Example 7). By operating the trap under force feedback control, the tension was maintained on the DNA at 10-22 pN, as indicated. Additional controls and considerations ascertained that the observed activity stemmed from a single Rep-X regardless of the 3′-tail length and inclusion/omission of SSB (Example 7). Remarkably, 95% (38 out of 40) of the Rep-X-DNA complexes tethered through a 3′-tail unwound the entire 6-kbp DNA in a processive manner (FIG. 3B, D) and the average pause-free speed was 136 bp/s (FIG. 3C). In comparison, only 3% (2 out of 61 at 4 pN tension, none at higher forces) of wild type Rep and 7% (5 out of 70) of Rep-Y complexes displayed such processive unwinding events (FIG. 3D). Rep-X may have even greater processivity than 6-kbp, currently only limited by the length of the DNA used. The processive activity of a crosslinked Rep-X monomer shows the innate potential of these helicases that can be harnessed via conformational control.

The amount of force Rep-_(X) can generate during unwinding was evaluated by performing measurements without the force feedback. Fixing trap positions led to a rapid build-up of force on the Rep-_(X) in the opposite direction of unwinding until the measurement was terminated due to the breakage of connection between the two beads (FIG. 3E). The highest loads achieved in this experiment were not enough to stall the helicase permanently. More detailed analysis showed that the pause free unwinding rate of Rep-_(X) was not impeded by increasing loads up to the limits of the linear regime of our trap (FIG. 3F), approximately 60 pN. These results suggest that the engineered Rep-X is the strongest helicase known to date (T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, S. M. Block, Forward and reverse motion of single RecBCD molecules on DNA. Biophysical journal 86, 1640-1648 (2004); J. G. Yodh, M. Schlierf, T. Ha, Insight into helicase mechanism and function revealed through single-molecule approaches. Quarterly reviews of biophysics 43, 185-217 (2010))

In order to test if generation of a super active helicase can be reproduced for other helicases, thereby providing additional evidence of the conformational control mechanism, a PcrA-X helicase was engineered from Bacillus stearothermophilus PcrA. The Rep mutant sequences used to generate PcrA-_(X) include those nucleotide and amino acid sequences identified in Table 3.

TABLE 3  Amino Acid and Nucleotide Sequences for exemplary PcrA-_(X) proteins Polypeptide/DNA/RNA (SEQ ID NO:_) 5′→3′ (nucleotide sequence) N→C (amino acid sequence) Wild type PcrA helicase ATGAACTTTTTATCGGAACAGCTGCTCGCCCATTTAAACAAAG (gene sequence) AGCAACAAGAAGCCGTCAGGACGACGGAAGGCCCGCTGCTCA >gi|696477066:c17795- TTATGGCGGGGGCGGGAAGCGGGAAAACGCGGGTGTTGACGC 15621 Geobacillus ACCGCATCGCCTATTTGATGGCGGAAAAGCATGTGGCGCCGT stearothermophilus GGAACATTTTGGCCATTACGTTTACGAACAAGGCGGCGCGCG ATCC 7953 AAATGCGGGPACGTGTGCAGTCGCTCTTAGGTGGGGCGGCGG GBScontig0000036_2, AAGACGTCTGGATTTCGACGTTCCACTCGATGTGCGTCCGCAT whole genome shotgun TTTGCGCCGCGACATTGACCGCATCGGCATCAACCGCAATTTT sequence (SEQ ID NO:  TCCATCCTTGATCCGACGGACCAGCTTTCAGTCATGAAAACGA 38) TTTTAAAAGAAAAAAACATAGACCCGAAAAAATTTGAGCCGC GGACGATTTTAGGCACGATCAGCGCGGCGAAAAACGAGCTGT TGCCTCCGGAGCAATTCGCGAAGCGGGCCTCGACGTATTACG AAAAAGTCGTCAGCGATGTGTATCAAGAATACCAACAGCGCC TGCTTCGCAATCATTCGCTCGATTTTGACGATTTGATCATGAC GACGATCCAACTGTTTGACCGCGTGCCGGATGTGCTTCACTAT TACCAATATAAGTTTCAGTACATTCATATTGATGAGTACCAGG ATACGAACCGCGCTCAATATACGCTGGTCAAAAAGCTGGCGG AACGCTTTCAAAACATTTGCGCCGTCGGCGACGCCGACCAAT CGATTTATCGGTGGCGCGGGGCGGACATCCAAAACATTTTGTC GTTCGAGCGCGACTATCCGAACGCAAAAGTCATTTTGCTTGAA CAAAACTACCGCTCGACGAAGCGCATTTTGCAAGCGGCGAAC GAAGTCATCGAGCATAACGTCAACCGGAAGCCGAAACGGCTT TGGACGGAAAACCCGGAAGGAAAGCCGATTCTTTATTATGAG GCGATGAACGAAGCGGACGAAGCGCAGTTTGTCGCTGGACGC ATCCGCGAGGCGGTGGAGCGCGGCGAACGCCGCTACCGTGAT TTTGCTGTCTTGTACCGGACGAACGCCCAGTCGCGTGTCATGG AGGAAATGTTGCTGAAAGCGAACATTCCGTATCAAATTGTCG GCGGCTTAAAGTTCTATGACCGGAAAGAAATTAAAGACATTC TCGCCTATTTGCGCGTCATTGCCAATCCGGACGATGATTTAAG CTTGCTTCGCATCATTAACGTGCCAAAACGCGGCATTGGCGCC TCGACGATCGACAAACTCGTCCGCTATGCAGCCGATCATGAG CTGTCCTTGTTTGAGGCGCTCGGCGAGCTAGAGATGATCGGGC TTGGCGCCAAAGCGGCCGGGGCGCTCGCCGCGTICCGCAGCC AGCTCGAGCAATGGACACAGCTGCAAGAATACGTCTCCGTCA CCGAACTCGTCGAAGAAGTGCTCGACAAATCGGGCTACCGCG AGATGCTCAAGGCGGAGCGGACGATTGAAGCACAAAGCCGG CTCGAGAACTTGGATGAGTTTTTGTCGGTGACGAAGCATTTTG AAAATGTGAGCGACGATAAATCGCTCATCGCCTTTTTAACCGA CTTGGCGCTCATTTCCGATTTGGACGAGCTGAACGGGACGGA ACAGGCCGCTGAAGGAGATGCCCGTCATGTTGATGACGTTGCA TGCCGCCAAAGGGCTCGAGTTTCCGGTCGTCTTTTTGATCGGC ATGGAAGAAGGCATTTTCCCGCACAACCGCTCTCTCGAGGAT GACGATGAGATGGAAGAAGAACGGCGGCTGGCGTACGTCGG CATCACCCGCGCGGAGGAAGAACTTGTGCTGACGAGCGCGCA AATGCGGACGTTGTTTGGCAACATCCAAATGAACCCGCCGTC GCGCTTTTTGAATGAAATTCCGGCGCATTTGCTTGAGACAGCC TCGCGCCGCCAAGCGGGCGCCTCCCGCCCGGCCGTTTCGCGCC CGCAGGCAAGCGGCGCCGTGGGATCGTGGAAAGTCGGCGATC GGGCGAATCACCGGAAATGGGGCATCGGCACCGTCGTCAGCG TCCGCGGCGGCGGCGACGACCAAGAGCTCGACATCGCCTTCC CGAGCCCGATCGGCATTAAACGGTTGCTTGCCAAATTTGCGCC GATTGAGAAAGTGTAG Wild type PcrA helicase MNFLSEQLLAHLNKEQQEAVRTTEGPLLIMAGAGSGKTRVLTHR (amino acid sequence) IAYLMAEKHVAPWNILAITFTNKAAREMRERVQSLLGGAAEDV >gi|696477065|ref| WISTHSMCVRILRRDIDRIGINRNFSILDPTDQLSVMKTILKEKNI WP_033016687.1 ATP- DPKKFEPRTILGTISAAKNELLPPEQFAKRASTYYEKVVSDVYQE dependent DNA helicase YQQRLLRNHSLDFDDLIMTTIQLFDRVPDVLHYYQYKYFQYIHIDE PcrA [Geobaciilus YQDTNRAQYTLVKKLAERFQNICAVGDADQSIYRWRGADIQNIL stearothermophilus] SFERDYPNAKVILLEQNYRSTKRILQAANEVIEHNVNRKPKRLWT (SEQ ID NO: 39) ENPEGKPILYYEAMNEADEAQFVAGRIREAVERGERRYRDFAVL YRTNAQSRVMEEMLLKANIPYQIVGGLKFYDRKEIKDILAYLRVI ANPDDDLSLLRIINVPKRGIGASTIDKLVRYAADHELSLFEALGEL EMIGLGAKAAGALAAFRSQLEQWTQLQEYVSVTELVEEVLDKS GYREMLKAERTIEAQSRLENLDEFLSVTKHFENVSDDKSLIAFLT DLALISDLDELNGTEQAAEGDAVMLMTLHAAKGLEFPVVFLIGM EEGIFPHNRSLEDDDEMEEERRLAYVGITRAEEELVLTSAQMRTL FGNIQMNPPSRFLNEIPAHLLETASRRQAGASRPAVSRPQASGAV GSWKVGDRANHRKWGIGTVVSVRGGGDDQELDIAFPSPIGIKKL LAKFAPIEKV PcrA-_(X) polypeptide¹ MNFLSEQLLAHLNKEQQEAVRTTEGPLLIMAGAGSGKTRVLTHR (SEQ ID NO: 9) IAYLMAEKHVAPWNILAITFTNKAAREMRERVQSLLGGAAEDV WISTFHSMAVRILRRDIDRIGINRNFSILDPTDQLSVMKTILKEKNI DPKKFEPRTILGTISAAKNELLPPEQFAKRASTYYEKVVSDVYQE YQQRLLRCHSLDFDDLIMTTIQLFDRVPDVLHYYQYKFQYIHIDE YQDTNRAQYTLVKKLAERFQNIAAVGDADQSIYRWRGADIQNIL SFERDYPNAKVILLEQNYRSTKRILQAANEVIEHNVNRKPKRLWT ENPEGKPILYYEAMNEADEAQFVAGRIREAVERGERRYRDFAVL YRTNAQSRVMEEMLLKANIPYQIVGGVKFYDRKEIKDILAYLRVI ANPDDDCSLLRIINVPKRGIGASTIDKLVRYAADHELSLFEALGEL EMIGLGAKAAGALAAFRSQLEQWTQLQEYVSVTELVEEVLDKS GYREMLKAERTIEAQSRLENLDEFLSVTKHFENVSDDKSLIAFLT DLALISDLDELNGTEQAAEGDAVMLMTLHAAKGLEFPVVFLIGM EEGIFPHNRSLEDDDEMEEERRLAYVGITRAEEELVLTSAQMRTL FGNIQMNPPSRFLNEIPAHLLETASRRQAGASRPAVSKPQASGAV GSWKVGDRANHRKWGIGTVVSVRGGGDDQELDIAFPSPIGIKRL LAKFAPIEKV PcrA-_(X) DNA² ATGAACTTTTTATCGGAACAGCTGCTCGCCCATTTAAACAAAG (SEQ ID NO: 10) AGCAACAAGAAGCCGTCAGGACGACGGAAGGCCCGCTGcrcA TTATGGCGGGGGCGGGAAGCGGGAAAACGCGGGTGTTGACGC ACCGCATCGCCTATTTGATGGCGGAAAAGCATGTGGCGCCGT GGAACATTTTGGCCATTACGTTTACGAACAAGGCGGCGCGCG AAATGCGGGAACGTGTGCAGTCGCTCTTAGGTGGGGCGGCGG AAGACGTCTGGATTTCGACGTTCCACTCGATGGCCGTCCGCAT TTTGCGCCGCGACATTGACCGCATCGGCATCAACCGCAATTTT TCCATCCTTGATCCGACGGACCAGCTTTCAGTCATGAAAACGA TTTTAAAAGAAAAAAACATAGACCCGAAAAAATTTGAGCCGC GGACGATTTTAGGCACGACAGCGCGGCGAAAAACGAGCTGT TGCCTCCGGAGCAATTCGCGAAGCGGGCCTCGACGTATTACG AAAAAGTCGTCAGCGATGTGTATCAAGAATACCAACAGCGCC TGCTTCGCTGTCATTCGCTCGATTTTGACGATTTGATCATGACG ACGATCCAACTGTTTGACCGCGTGCCGGATGTGCTTCACTATT ACCAATATAAGTTTCAGTACATTCATATTGATGAGTACCAGGA TACGAACCGCGCTCAATATACGCTGGTCAAAAAGCTGGCGGA ACGCTTCAAAACATTGCCGCCGTCGGCGACGCCGACCAATC GATTTATCGGTGGCGCGGGGCGGACATCCAAAACATTTTGTC GTTCGAGCGCGACTATCCGAACGCAAAAGTCATTTTGCTTGAA CAAAACTACCGCTCGACGAAGCGCATTTTGCAAGCGGCGAAC GAAGTCATCGAGCATAACGTCAACCGGAAGCCGAAACGGCTT TGGACGGAAAACCCGGAAGGAAAGCCGATTCTTTATTATGAG GCGATGAACGAAGCGGACGAAGCGCAGTTTGTCGCTGGACGC ATCCGCGAGGCGGTGGAGCGCGGCGAACGCCGCTACCGTGAT TTTGCTGTCTTGTACCGGACGAACGCCCAGTCGCGTGTCATGG AGGAAATGTTGCTGAAAGCGAACATTCCGTATCAAATTGTCG GCGGCGTAAAGTTCTATGACCGGAAAGAAATTAAAGACATTC TCGCCTATTTGCGCGTCATTGCCAATCCGGACGATGATTGCAG CTTGCTTCGCATCATTAACGTGCCAAAACGCGGCATTGGCGCC TCGACGATCGACAAACTCGTCCGCTATGCAGCCGATCATGAG CTGTCCTTGTTTGAGGCGCTCGGCGAGCTAGAGATGATCGGGC TTGGCGCCAAAGCGGCCGGGGCGCTCGCCGCGTTCCGCAGCC AGCTCGAGCAATGGACACAGCTGCAAGAATACGTCTCCGTCA CCGAACTCGTCGAAGAAGTGCTCGACAAATCGGGCTACCGCG AGATGCTCAAGGCGGAGCGGACGATTGAAGCACAAAGCCGG CTCGAGAACTTGGATGAGTTTTTGTCGGTGACGAAGCATTTTG AAAATGTGAGCGACGATAAATCGCTCATCGCCTTTTTAACCGA CTTGGCGCTCATTTCCGATTTGGACGAGCTGAACGGGACGGA ACAGGCCGCTGAAGGAGATGCCGTCATGTTGATGACGTTGCA TGCCGCCAAAGGGCTCGAGTTTCCGGTCGTCTTTTTGATCGGC ATGGAAGAAGGCATTTTCCCGCACAACCGCTCTCTCGAGGAT GACGATGAGATGGAAGAAGAACGGCGGCTGGCGTACGTCGG CATCACCCGCGCGGAGGAAGAACTTGTGCTGACGAGCGCGCA AATGCGGACGTTGTTTGGCAACATCCAAATGAACCCGCCGTC GCGCTTTTTGAATGAAATTCCGGCGCATTTGCTTGAGACAGCC TCGCGCCGCCAAGCGGGCGCCTCCCGCCCGGCCGTTTCGCGCC CGCAGGCAAGCGGCGCCGTGGGATCGTGGAAAGTCGGCGATC GGGCGAATCACCGGAAATGGCUGCATCGGCACCGTCGTCAGCG TCCGCGGCGGCGGCGACGACCAAGAGCTCGACATCGCCTTCC CGAGCCCGATCGGUATTAAACGGTTGCTTGCCAAATTTGCGCC GATTGAGAAAGTGTAG PcrA-_(X) RNA³ AUGAACUUUUUAUCGGAACAGCUGCUCGCCCAUUUAAACAA (SEQ ID NO: 11) AGAGCAACAAGAAGCCGUCAGGACGACGGAAGGCCCGCUGC UCAUUAUGGCGGGGGCGGGAAGCGGGAAAACGCGGGUGUU GACGCACCGCAUCGCCUAUUUGAUGGCGGAAAAGCAUGUGG CGCCGUGGAACAUUUUGGCCAUUACGUUUACGAACAAGGCG GCGCGCGAAAUGCGGGAACGUGUGCAGUCGCUCUUAGGUGG GGCGGCGGAAGACGUCUGGAUUUCGACGUUCCACUCGAUGG CCGUCCGCAUUUUGCGCCGCGACAUUGACCGCAUCGGCAUC AACCGCAAUUUUUCCAUCCUUGAUCCGACGGACCAGCUUUC AGUCAUGAAAACGAUUUUAAAAGAAAAAAACAUAGACCCG AAAAAAUUUGAGCCGCGGACGAUUUUAGGCACGAUCAGCGC GGCGAAAAACGAGCUGUUGCCUCCGGAGCAAUUCGCGAAGC GGGCCUCGACGUAUUACGAAAAAGUCGUCAGCGAUGUGUAU CAAGAAUACCAACAGCGCCUGCUUCGCUGUCAUUCGCUCGA UUUUGACGAUUUGAUCAUGACGACGAUCCAACUGUUUGACC GCGUGCCGGAUGUGCUUCACUAUUACCAAUNUAAGUUUCAG UACAUUCAUAUUGAUGAGUACCAGGAUACGAACCGCGCUCA AUAUACGCUGGUCAAAAAGCUGGCGGAACGCUUUCAAAACA UUGCCGCCGUCGGCGACGCCGACCAAUCGAUUUAUCGGUGG CGCGGGGCGGACAUCCAAAACAUUUGUCGUUCGAGCGCGA CUAUCCGAACGCAAAAGUCAUUUUGCUUGAACAAAACUACC GCUCGACGAAGCGCAUUUUGCAAGCGGCGAACGAAGUCAUC GAGCAUAACGUCAACCGGAAGCCGAAACGGCUUUGGACGGA AAACCCGGAAGGAAAGCCGAUUCUUUAUUAUGAGGCGAUGA ACGAAGCGGACGAAGCGCAGUUUGUCGCUGGACGCAUCCGC GAGGCGGUGGAGCGCGGCGAACGCCGCUACCGUGAUUUUGC UGUCUUGUACCGGACGAACGCCCAGUCGCGUGUCAUGGAGG AAAUGUUGCUGAAAGCGAACAUUCCGUAUCAAAUUGUCGGC GGCGUAAAGUUCUAUGACCGGAAAGAAAUUAAAGACAUUC UCGCCUAUUUGCGCGUCAUUGCCAAUCCGGACGAUGAUUGC AGCUUGCUUCGCAUCAUUAACGUGCCAAAACGCGGCAUUGG CGCCUCGACGAUCGACAAACUCGUCCGCUAUGCAGCCGAUC AUGAGCUGUCCUUGUUUGAGGCGCUCGGCGAGCUAGAGAUG AUCGGGCUUGGCGCCAAAGCGGCCGGGGCGCUCGCCGCGUU CCGCAGCCAGCUCGAGCAAUGGACACAGCUGCAAGAAUACG UCUCCGUCACCGAACUCGUCGAAGAAGUGCUCGACAAAUCG GGCUACCGCGAGAUGCUCAAGGCGGAGCGGACGAUUGAAGC ACAAAGCCGGCUCGAGAACUUGGAUGAGUUUUUGUCGGUGA CGAAGCAUUUUGAAAAUGUGAGCGACGAUAAAUCGCUCAUC GCCUUUUUAACCGACUUGGCGCUCAUUUCCGAUUUGGACGA GCUGAACGGGACGGAACAGGCCGCUGAAGGAGAUGCCGUCA UGUUGAUGACGUUGCAUGCCGCCAAAGGGCUCGAGUUUCCG GUCGUCUUUUUGAUCGGCAUGGAAGAAGGCAUUUUCCCGCA CAACCGCUCUCUCGAGGAUGACGAUGAGAUGGAAGAAGAAC GGCGGCUGGCGUACGUCGGCAUCACCCGCGCGGAGGAAGAA CUUGUGCUGACGAGCGCGCAAAUGCGGACGUUGUUUGGCAA CAUCCAAAUGAACCCGCCGUCGCGCUUUUUGAAUGAAAUUC CGGCGCAUUUGCUUGAGACAGCCUCGCGCCGCCAAGCGGGC GCCUCCCGCCCGGCCGUUUCGCGCCCGCAGUCAAGCGGCGCC GUGGGAUCGUGGAAAGUCGGCGAUCGGGCGAAUCACCGGAA AUGUGGCAUCGGCACCGUCGUCAGCGUCCGCGGCGGCGGCG ACGACCAAGAGCUCGACAUCGCCUUCCCGAGCCCGAUCGGC AUUAAACGGUUGCUUGCCAAAUUUGCGCCGAUUGAGAAAGU GUAG PerA-_(X) polypeptide⁴ SEQ ID NO: 9 and formula no 1 in Table 2  (SEQ ID NO: 12) (1-[2-[2-[2-(2,5-dioxopyrrol-1- yl)ethoxy]ethoxy]ethyl]pyrrole-2,5-dime). ¹This PcrA mutant encodes mutations removing natural cysteine residues found in the wild-type PcrA and include further amino acid mutations to facilitate intramolecular crosslinking to an intramolecular crosslinking agent to generate the PcrA-_(X) polypeptide. ²The DNA sequence corresponds to the open reading frame that encodes the polypeptide of SEQ ID NO: 9. ³The RNA sequence corresponds to the open reading frame that encodes the polypeptide of SEQ ID NO: 9. ⁴The PcrA-_(X) polypeptide closed form monomer following reaction of PcrA-_(X) polypeptide (SEQ ID NO: 9) with an intramolecular crosslinking agent.

Exemplary intramolecular crosslinking agents suitable for generating versions of PcrA-_(X) include those identified in Table 2. Methods and conditions for generating intramolecular crosslink formation in proteins are well known in the art for these types of intramolecular crosslinking agents, and such methods and conditions are applicable to the PcrA helicases of this disclosure.

Mutations involved replacing two highly conserved Cys residues in this helicase (FIG. 4A, B) which reduced the apparent ssDNA-dependent ATPase activity from approximately 40 ATP/s (wild type) to 5 ATP/s. Upon crosslinking in the closed form, PcrA-_(X) retained the low ATPase activity (4.3 ATP/s), but exhibited an enhanced helicase activity in comparison to both the wild type and non-crosslinked mutant in ensemble reactions (FIG. 5A, B). smFRET experiments showed that PcrA-X monomers can unwind 39% (228 out of 578) of the 18-bp dsDNA they bind compared to only 4% (26 out of 617) for wild type PcrA (FIG. 6A-C). In the optical tweezers assay, PcrA-X monomers, like Rep-X, were capable of processively unwinding of 1-6 kbp long DNA, albeit at a much lower rate (2-15 bp/s, FIG. 6D) whereas no PcrA molecule (0 out of 51) was able to do the same (FIG. 6E). Despite the impaired activity levels of the PcrA mutant, conversion to PcrA-X made its monomers into highly processive helicases, thus indicating a general mechanism of conformational control for this class of helicases.

Strong helicase activity of Rep-X and PcrA-X raises the possibility that the cellular partners of Rep or PcrA may switch on the powerful unwinding activity intrinsic to these enzymes by constraining them in the closed conformation. One such partner of PcrA is RepD, a plasmid replication initiator protein from Staphylococcus aureus that recognizes and forms a covalent adduct with the oriD sequence of the plasmid, and then recruits PcrA for highly processive unwinding (A. F. Slatter, C. D. Thomas, M. R. Webb, PcrA helicase tightly couples ATP hydrolysis to unwinding double-stranded DNA, modulated by the initiator protein for plasmid replication, RepD. Biochemistry 48, 6326-6334 (2009); W. Zhang et al., Directional loading and stimulation of PcrA helicase by the replication initiator protein RepD. Journal of molecular biology 371, 336-348 (2007); C. Machon et al., RepD-mediated recruitment of PcrA helicase at the Staphylococcus aureus pC221 plasmid replication origin, oriD. Nucleic acids research 38, 1874-1888 (2010)). Based on the similar results from PcrA-X and the homologous E. coli counterpart Rep-X, but not Rep-Y, we hypothesized that the RepD-induced PcrA activity enhancement is in fact the result of the conformational constraint of the helicase in the PcrA-X-like closed form. To test this prediction, we prepared an oriD DNA-RepD adduct, and measured the intramolecular conformation of PcrA bound to this adduct. We used a double cysteine mutant of PcrA, PcrA-DM1, stochastically labeled with a mixture of donor and acceptor fluorophores that would be expected to generate high E_(FRET) in the closed form and low E_(FRET) in the open form (J. Park et al., PcrA helicase dismantles RecA filaments by reeling in DNA in uniform steps. Cell 142, 544-555 (2010); (FIG. 6F). The E_(FRET) distributions of PcrA-DM1 bound to the oriD DNA-RepD adduct and the oriD DNA alone are shown in FIG. 6F. Only the PcrA-DM1 molecules with a fluorescence active Cy3-Cy5 pair were included in the analysis. The results revealed that the presence of RepD indeed biases PcrA toward the closed high E_(FRET) conformation. Without the invention being limited to any particular mechanism, the regulation mechanism of this class of helicases may involve in vivo partner proteins that constrain the conformation of 2B subdomain to the closed form to activate its function.

The basis for constraining Rep and PcrA into the closed form that converts an enzyme with undetectable unwinding activity to a super helicase is unknown. One possibility is that the intrinsic unwinding activity itself requires the closed form, for example via the torque-wrench mechanism proposed for UvrD (J. Y. Lee, W. Yang, UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke. Cell 127, 1349-1360 (2006)). Another possibility is that the open form inhibits helicase function and crosslinking to the closed form prevents this inhibitory mechanism. Without the invention being limited to any particular theory of operation, we prefer the latter for the following reasons. First, Rep-Y crosslinked in the open form does unwind DNA as well as the wild type when the protein is at high concentrations in excess of DNA (FIG. 1E). Therefore, the closed form per se is not absolutely required for unwinding activity. Second, using ultra-high resolution optical tweezers combined with smFRET capability, we found that UvrD assumes the closed conformation when it unwinds DNA but after it unwinds about 10 bp it switches to the open conformation and rewinds the DNA likely after strand switching. Therefore, we suggest that Rep-X becomes a highly processive super-helicase because crosslinking prevents the open conformation required for strand-switching and rewinding that have been observed for UvrD (M. N. Dessinges, T. Lionnet, X. G. Xi, D. Bensimon, V. Croquette, Single-molecule assay reveals strand switching and enhanced processivity of UvrD. Proc. Natl. Acad. Sci., U.S.A. 101, 6439-6444 (2004)) and BLM (J. G. Yodh, B. C. Stevens, R. Kanagaraj, P. Janscak, T. Ha, BLM helicase measures DNA unwound before switching strands and hRPA promotes unwinding reinitiation. The EMBO journal 28, 405-416 (2009)). The enhancement of unwinding activity via the deletion of 2B domain in Rep (W. Cheng et al., The 2B domain of the Escherichia coli Rep protein is not required for DNA helicase activity. Proc. Natl. Acad. Sci., U.S.A. 99, 16006-16011 (2002)) may also be due to inhibition of strand switching (M. J. Comstock, K. D. Whitley, H. Jia, T. M. Lohman, T. Ha and Y. R. Chemla, “Direct observation of structure-function relationship in a nucleic acid processing enzyme,” Science 348: 352-354 (2015).

Most conformational control of protein functions demonstrated so far first locks the naturally active protein to an artificially inhibited conformation so that additional controls imposed by researchers can be used to recover the original activity (B. Choi, G. Zocchi, Y. Wu, S. Chan, L. Jeanne Perry, Allosteric control through mechanical tension. Phy Rev Lett 95, 078102 (2005); M. Tomishige, R. D. Vale, Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change. J Cell Biol 151, 1081-1092 (2000); D. M. Veine, K. Ohnishi, C. H. Williams, Jr., Thioredoxin reductase from Escherichia coli: evidence of restriction to a single conformation upon formation of a crosslink between engineered cysteines. Protein science: a publication of the Protein Society 7, 369-375 (1998); B. X. Huang H. Y. Kim, Interdomain conformational changes in Akt activation revealed by chemical cross-linking and tandem mass spectrometry. Mol Cell Proteomics 5, 1045-1053 (2006)). Our work is innovative and unique in that we found a conformational control that activates a naturally inhibited unwinding function, and the resulting enzyme is a super-helicase that has unprecedentedly high processivity for a single motor helicase. RecBCD, another SF-1 helicase, has similarly high processivity but contains two motors and associated nucleases. Moreover it is known to backslide at opposing forces below 10 pN whereas Rep-X can be active against forces as high as 60 pN (Perkins et al (2004) supra). This super helicase with high processivity and high tolerance against load without nuclease activities may also be useful for biotechnological applications such as single molecule nanopore sequencing (D. Branton et al., The potential and challenges of nanopore sequencing. Nature biotechnology 26, 1146-1153 (2008); A. H. Laszlo et al., Decoding long nanopore sequencing reads of natural DNA. Nature biotechnology, (2014)) and isothermal DNA amplification (M. Vincent, Y. Xu, H. Kong, Helicase-dependent isothermal DNA amplification. EMBO reports 5, 795-800 (2004).

In this regard, one type of isothermal DNA amplification for Which these super helicases have application include helicase dependent amplification. Referring to FIG. 8, the helicase dependent amplification can be characterized in three steps. In step 1, DNA helicase (104) contacts a double-stranded DNA (101) to unwind the first and second single strands (102 and 103) to provide the ability of first and second oligonucleotide primers (105 and 106) hybridize to the first and second single strands (102 and 103), respectively. In step 2: DNA-dependent DNA polymerases (107) bind to the 3′-termini of the first and second oligonucleotide primers (105 and 106) to initiate chain elongation of new strands (108 and 109). In step 3, continued DNA polymerization results in DNA amplification and formation of new double-stranded DNA (110 and 111).

Nucleic Acid Amplification

In certain exemplary embodiments, methods for amplifying nucleic acid sequences are provided. Exemplary methods for amplifying nucleic acids include the polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263 and Cleary et al. (2004) Nature Methods 1:241; and U.S. Pat. Nos. 4,683,195 and 4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:360-364), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:1874), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:1173), Q-Beta Replicase (Lizardi et at (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000) J. Biol. Chem. 275:2619; and Williams et al. (2002) J. Biol. Chem. 277:7790), the amplification methods described in U.S. Pat. Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199, isothermal amplification (e.g., rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HDA), PWGA, or any other nucleic acid amplification method using techniques well known to those of skill in the art.

“Polymerase chain reaction,” or “PCR,” refers to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art, e.g., exemplified by the references: McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in a conventional PCR using Taq DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature greater than 90° C., primers annealed at a temperature in the range 50-75° C., and primers extended at a temperature in the range 72-78° C.

The term “PCR” encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR and the like. Reaction volumes range from a few hundred nanoliters, e.g., 200 nL, to a few hundred microliters, e.g., 200 microliters. “Reverse transcription PCR,” or “RT-PCR,” means a PCR that is preceded by a reverse transcription reaction that converts a target RNA to a complementary single stranded DNA, which is then amplified, e.g., Tecott et al., U.S. Pat. No. 5,168,038. “Real-time PCR” means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., Gelfand et al., U.S. Pat. No. 5,210,015 (“Taqman”); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002). “Nested PCR” means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. As used herein, “initial primers” in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and “secondary primers” mean the one or more primers used to generate a second, or nested, amplicon. “Multiplexed PCR” means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture, e.g. Bernard et al. (1999) Anal. Biochem., 273:221-228 (two-color real-time PCR). Usually, distinct sets of primers are employed for each sequence being amplified. “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Techniques for quantitative PCR are well-known to those of ordinary skill in the art, as exemplified in the following references: Freeman et al., Biotechniques, 26:112-126 (1999); Becker-Andre et al., Nucleic Acids Research, 17:9437-9447 (1989); Zimmerman et al., Biotechniques, 21:268-279 (1996); Diviacco et al., Gene, 122:3013-3020 (1992); Becker-Andre et al., Nucleic Acids Research, 17:9437-9446 (1989); and the like.

In one aspect of the invention, a method of performing isothermal DNA amplification is provided. The method can includes two steps. The first step includes forming a mixture. The mixture includes a double-stranded DNA template having a first strand and a second strand; a conformationally-constrained helicase; a DNA-dependent DNA polymerase; a first oligonucleotide primer complementary to a portion of the first strand; a second oligonucleotide primer complementary to a portion of the second strand; and an amplification buffer cocktail. The second step includes incubating the mixture at a temperature compatible for activating the conformationally-constrained helicase and DNA-dependent DNA polymerase. In some embodiments of this aspect, the conformationally-constrained helicase is selected from SEQ ID NOs: 4 and 12.

Nucleic Acid Sequencing

In certain exemplary embodiments, methods of determining the sequence identities of nucleic acid sequences are provided. Determination of the sequence of a nucleic acid sequence of interest can be performed using variety of sequencing methods known in the art including, but not limited to, sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescent in situ sequencing (FISSEQ), FISSEQ beads (U.S. Pat. No. 7,425,431), wobble sequencing (PCT/US05/27695), multiplex sequencing (U.S. 2008/0269068; Porreca et al (2007) Nat. Methods 4:931), polymerized colony (POLONY) sequencing (U.S. Pat. Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US05/06425), nanogrid rolling circle sequencing (ROLONY) (U.S. 2009/0018024), nanopore sequencing (using platforms such as those from Agilent, Oxford, Sequenom, Noblegen, NABsys, Genia), allele-specific oligo ligation assays (e.g., oligo ligation assay (OLA), single template molecule OLA using a ligated linear probe and a rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using a ligated circular padlock probe and a rolling circle amplification (RCA) readout) and the like. High-throughput sequencing methods, e.g., on cyclic array sequencing using platforms such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrents, Complete Genomics, Pacific Bioscience, Helicos, Polonator platforms (Worldwide Web Site: Polonator.org), and the like, can also be utilized. High-throughput sequencing methods are described in U.S. 2010/0273164. A variety of light-based sequencing technologies are known in the art (Landegren et at (1998) Genome Res. 8:769-76; Kwok (2000) Pharmocogenomics 1:95-100; and Shi (2001) Clin. Chem. 47:164-172).

In certain exemplary embodiments, the DNA-dependent DNA polymerase is selected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol I Large Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment Bsu DNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase and phi29 DNA Polymerase. In some embodiments, the conformationally-constrained helicase includes a helicase selected from superfamily 1, wherein the helicase has a first amino acid residue and a second amino acid reside, and wherein the first and second amino acid residues are in proximity. The conformationally-constrained helicase also includes a linker, wherein the linker comprises a first covalent bond with the first amino acid residue and a second covalent bond with the second amino acid residue. In some embodiments of this aspect, the conformationally-constrained helicase includes a crosslinked, closed form helicase monomer.

Expression of Helicase-_(X) Polypeptides

The nucleic acids encoding the Rep-_(X) and PcrA-_(X) polypeptides can be adapted to suitable expression systems for producing the helicase_(X) polypeptides for helicase-_(X) production. For DNAs encoding helicase_(X) genes, the representative genes can be operably-linked to suitable expression vectors for expressing the proteins in bacterial, fungal, insect or other suitable expression host. For RNAs encoding helicase-_(X) polypeptides, the representative RNAs can be engineered for enabling efficient expression in vitro of the polypeptides in extract lysates produced from bacterial, fungal, insect or other suitable expression host sources. Such systems are well known in the art. Following expression, the helicase-_(X) polypeptides can be purified by methods known in the art, including affinity-tag chromatography, SDS-PAGE, and size-exclusion chromatography, among others.

In certain exemplary embodiments, vectors such as, for example, expression vectors, containing a nucleic acid encoding one or more helicase-_(X) polypeptides described herein are provided. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

In certain exemplary embodiments, the recombinant expression vectors comprise a nucleic acid sequence (e.g., a nucleic acid sequence encoding one or more helicase-_(X) polypeptides described herein) in a form suitable for expression of the nucleic acid sequence in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence encoding one or more helicase-_(X) polypeptides is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors described herein can be introduced into host cells to thereby produce proteins or portions thereof, including fusion proteins or portions thereof, encoded by nucleic acids as described herein (e.g., one or more helicase_(X) polypeptides).

Recombinant expression vectors of the invention can be designed for expression of one or more encoding one or more helicase-_(X) polypeptides in prokaryotic or eukaryotic cells. For example, one or more vectors encoding one or more helicase-_(X) polypeptides can be expressed in bacterial cells such as E. coli, insect cells (e.g., using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40); pMAL (New England Biolabs, Beverly, Mass.); and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector encoding one or more helicase-_(X) polypeptides is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et. al., (1987) EMBO J. 6:229-234); pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943); pJRY88 (Schultz et al., (1987) Gene 54:113-123); pYES2 (Invitrogen Corporation, San Diego, Calif.); and picZ (Invitrogen Corporation).

Alternatively, one or more helicase-_(X) polypeptides can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In certain exemplary embodiments, a nucleic acid described herein is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see Green M., and Sambrook; J. Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012.

In certain exemplary embodiments, host cells into which a recombinant expression vector of the invention has been introduced are provided. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, one or more helicase-_(X) polypeptides can be expressed in bacterial cells such as E. coli, viral cells such as retroviral cells, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

Delivery of nucleic acids described herein (e.g., vector DNA) can be by any suitable method in the art. For example, delivery may be by injection, gene gun, by application of the nucleic acid in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, or by any other suitable transfection method.

As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTIN™ (Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE™ (Invitrogen), FUGENE™ (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, N.Y.), EFFECTENE™ (Qiagen, Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France) and the like), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Green and Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012), and other laboratory manuals.

Kits

In another aspect, kits are contemplated in this disclosure. For example, a kit for performing helicase dependent amplification is provided. The kit can include a conformationally-constrained helicase and an optional amplification butler cocktail. The conformationally-constrained helicase of the kit includes one or more helicase_(X) polypeptides having a covalent linkage (e.g., reacted with a suitable intramolecular crosslinking agent) to form closed form helicase-_(X) monomers having super helicase activity of the type described for Rep-X and PcrA-X. In particular, the conformationally-constrained helicase can be generated form reacting SEQ ID NOs:4 and 9 with a suitable intramolecular crosslinking agent. Representative conformationally-constrained helicases include those of SEQ ID NOs:4 and 12.

The kit can further include a DNA-dependent DNA polymerase. Exemplary DNA-dependent DNA polymerases for inclusion in kit include a polymerase selected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol I Large Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment, Bsu DNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase, phi29 DNA Polymerase and the like.

EXAMPLES Example 1 Mutagenesis and Purification of Protein

Preparation of pET expression plasmids containing cysteine-less rep (C18L, C43S, C167V, C178A, C612A) and pcrA (C96A/C247A) with N-terminal hexa-histidine-tags (SEQ ID NO: 36) were pet formed as described previously (Park et al (2005) supra, I. Rasnik, S. Myong, W. Cheng T. M. Lohman, T. Ha, DNA-binding orientation and domain conformation of the E. coli rep helicase monomer bound to a partial duplex junction: single-molecule studies of fluorescently labeled enzymes. J. Mol. Biol. 336, 395-408 (2004)). Site-directed mutations to introduce two Cys residues for crosslinking (Rep-X: A178C/S400C, Cys178 is a native cysteine in the wild type, Rep-Y: D127C/S494C, PcrA-X: N187C/L409C) were done using QuikChange Lightning kit (Life Technologies, Inc.) and mutagenic primer oligonucleotides (Integrated DNA Technologies Inc., Coralville, Iowa). Protein purifications were performed as described previously (Park et al. (2005) supra; Rasnik et al (2004) supra). Catalytic activity levels of purified proteins as well as those of the crosslinked samples were determined in a ssDNA-dependent ATPase activity assay using the Invitrogen EnzChek phosphate assay kit (Life Technologies Inc.), the oligonucleotide (dT)₄₅ (SEQ ID NO: 305) and 1 mM ATP in buffer D (see ensemble FRET unwinding assay).

Wild type RepD from Staphylococcus aureus was purified as described in (Slatter et al. ((2009) supra; Zhang et al., (2007) supra) with the following differences. A wt-RepD encoding pET11m-RepD plasmid was constructed for expression in B834 (pLysS). The gene sequence contained silent mutations to introduce restriction sites for AgeI, PstI, SacI, and to modify the nick site (TCTAAT to TCGAAT) to prevent premature cleavage by RepD during expression. An ammonium sulfate precipitated pellet (from 0.5 L culture) was resuspended and run through serially connected 5 ml Q-Sepharose (removed once the sample was through) and 5 ml heparin-Sepharose cartridges connected in series (GE Healthcare), and eluted on an ÄKTA purifier 10 FPLC system.

Example 2 Intra-crosslinking of Rep and PcrA

Dual-cysteine Rep mutants were incubated overnight at 4° C. with 2- to 100-fold excess of bis-maleimide crosslinkers DTME (13 Å) and BMOE (8 Å) purchased from Thermo Fisher Scientific, Rockford, Ill. (FIG. 10). PcrA-X was crosslinked with DTME and BM(PEG)2 (14.7 Å) from the same manufacturer. Excess crosslinkers were removed by Bio-Rad P-30 desalting column. Crosslinked Rep-X, Rep-Y and PcrA-X samples were stored at −20° C. or −80° C. as described (Park et al. (2005) supra; Rasnik et al. (2004) supra). Data presented in this manuscript used BMOE (8 Å), but other crosslinkers of various lengths gave similar results. DTME is a di-sulfide containing crosslinker that we reduced with β-mercaptoethanol (β-ME) or tris(2-carboxyethyl) phosphine (TCEP) to revert the crosslinked helicase to the non-crosslinked form for control purposes.

Crosslinking of the double Cys mutants with the his-maleimide linkers has the potential of producing covalently attached multimeric species, in addition to the intended internally crosslinked monomeric species. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can distinguish these species from the non-crosslinked monomers (I. L. Urbatsch et al., Cysteines 431 and 1074 are responsible for inhibitory disulfide cross-linking between the two nucleotide-binding sites in human P-glycoprotein. J. Biol. Chem, 276, 26980-26987 (2001)). Here we show a representative analysis of a crosslinked Rep-Y sample. Crosslinked Rep-X and Rep-Y produced three bands on a SDS polyacrylamide gel (FIG. 7A): a bottom band at ˜76 kDa that was the same as the non-crosslinked Rep, a slightly retarded dominant middle band at ˜100 kDa for Rep-Y and ˜90 kDa for Rep-X and a much more slowly migrated, very faint top band at ˜300 kDa. FIG. 7B shows three such bands of a Rep-Y sample (lane Rep-Y) crosslinked with a cleavable di-sulfide containing crosslinker (DTME). The dominant middle band and the faint top band were the crosslinked species because they disappeared upon cleavage of the crosslinker using beta-mercaptoethanol (β-ME) (lane Rep-Y*). Relative shift between the middle bands of Rep-X and Rep-Y (FIG. 7A) was a strong indication of an internally crosslinked monomeric species, because the denatured Rep-X and Rep-Y would be likely to migrate at different rates due to the different size of peptide loops introduced by the internal crosslinker (denatured Rep-Y has a loop of 368 amino acids (aa) whereas Rep-X loop is 223 aa long). In order to ensure that the dominant middle band is not multimeric but is the intramolecularly crosslinked monomeric species, a Rep-Y sample was fractionated according to molecular size on a Superdex 200 size exclusion chromatography (SEC) column controlled by an FPLC apparatus. Elution profiles of Rep-Y and non-crosslinked Rep are shown in the FIG. 7C. Eluted fractions were analyzed on an SDS polyacrylamide gel (FIG. 7D, lanes F1-F7). The multimeric species that was eluted in the early SEC fractions (11-13 ml) displayed only the top band whereas the dominant middle band was eluted together with the non-crosslinked Rep monomer in the SEC analysis, showing that the middle band represents the intramolecularly crosslinked species and the top band is multimeric. After establishing that the intra-crosslinked protein shows up as a retarded band compared to the non-crosslinked form on the SDS polyacrylamide gels (such as the Rep-Y data presented here), we used this assay to check the efficiency of crosslinking reactions for Rep-X, Rep-Y and PcrA-X (86%, 73% and 58% respectively for the samples used in this manuscript). The Rep-Y form exhibited ATPase activity on par with non-crosslinked Rep (FIG. 7E).

Example 3 Size Exclusion Chromatography and SDS-PAGE Analysis

Crosslinked Rep and PcrA samples were separated from multimeric byproducts using Superdex 200 grade 10/300GL or HiLoad 16/600 gel filtration columns on an ÄKTA purifier 10 FPLC system. The crosslinking efficiency was monitored by SDS-PAGE analysis on 7.5-10% Tris-glycine gels (Bio-Rad). As needed for gel analysis, reduction of samples crosslinked with DTME was achieved by adding 5% (v/v) β-ME during the SDS denaturation step.

Example 4 Ensemble FRET Unwinding Assay

Multiple turnover ensemble unwinding kinetics was used to gauge the effect of the mutations and conformational modifications to the helicase activity. We used an 18-bp FRET labeled DNA substrate with a 3′-(dT)₁₀ overhang (SEQ ID NO: 33) (FIG. 1C), constructed by annealing complementary oligonucleotides DNA7 (Cy5-GCC TCG CTG CCG TCG CCA (SEQ ID NO: 40)) and amino-dT labeled DNA8 (TGG CGA CGG CAG CGA GGC-(T-Cy3)-T₁₀ (SEQ ID NO: 41)). Alternatively, another similarly labeled 50-bp DNA with 3′-(dT)₃₀ overhang (SEQ ID NO: 17) was also used. This construct was made by annealing oligonucleotides DNA9 (Cy5-TCA ACT AGC AGT CAT AGG AGA AGT ATT AAC ATG CCT CGC TGC CGT CGC CA (SEQ ID NO: 42)) and amino-dT labeled DNA10 (TG GCG ACG GCA GCG AGG CAT GTT AAT ACT TCT CCT ATG ACT GCT AGT TGA (T-Cy3) T₂₉ (SEQ ID NO: 43)). Unless otherwise stated, 5 nM ensemble FRET DNA was mixed with 50 nM helicase in buffer D (10 mM Tris-HCl [pH 8.0], 15 mM NaCl, 10 mM MgCl₂, 10% (v/v) glycerol, 0.1 mg/ml BSA) and 1 mM ATP was added to start the unwinding reaction in a quartz cuvette. A Cary Eclipse fluorescence spectrophotometer was used to measure the donor (I_(555nm)) and the acceptor signals (I_(667nm)) under 545-nm excitation (5-nm slit, 2-10 Hz acquisition rate and 600-900V photomultiplier voltage). Unwinding of the substrate was monitored by the decrease in ensemble E_(FRET) value, defined as E_(FRET-ensemble)=I_(667nm)/(I_(555nm)−I₀+I_(667nm)) where I₀ was the baseline donor signal of unpaired Cy3 prior to addition of ATP.

Example 5 smFRET Unwinding and RepD-PcrA Interaction Assays

All smFRET experiments were conducted on a custom-built prism type TIRF microscopy stage with an Andor EMCCD camera as described in R. Roy, S. Hohng, T. Ha, A practical guide to single-molecule FRET. Nat Methods 5, 507-516 (2008) and C. Joo, T. Ha, in Cold Spring Harb Protoc. (2012), vol. 2012. Reaction chambers were formed by quartz slides and glass coverslips passivated with polyethyleneglycol (PEG) and 1% biotinylated PEG (mPEG-SC and bio-PEG-SC, Laysan Bio, Arab, Ala.), followed by 5 min incubation with Neutravidin (Thermo Scientific, Newington, N.H.) for immobilization of biotinylated molecules on the chamber surface as described below.

For the smFRET unwinding experiments, the reaction chamber was first incubated with biotinylated anti penta-histidine tag (SEQ ID NO: 44) antibody (Qiagen, Valencia, Calif.), followed by 10-30 min incubation of His₆-tagged (SEQ ID NO: 36) helicase sample (0.5-1 nM). The unwinding of the DNA was initiated by flowing 1 nM smFRET DNA and 1 mM ATP in the reaction buffer A (10 mM Tris-HCl [pH 8.0], 10 mM MgCl₂, 15 mM NaCl, 10% (v/v) glycerol, 1% (v/v) gloxy and 0.2% (w/v) glucose, an oxygen scavenging system (Y. Harada, K. Sakurada, T. Aoki, D. D. Thomas, T. Yanagida, Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay. J. Mol. Biol. 216, 49-68 (1990).) and 3-4 mM Trolox (T. Yanagida, M. Nakase, K. Nishiyama, F. Oosawa, Direct observation of motion of single F-actin filaments in the presence of myosin. Nature 307, 58-60 (1984); I. Rasnik, S. A. McKinney, T. Ha, Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3, 891-893 (2006)). The smFRET DNA substrate was constructed by annealing the oligonucleotides DNA3 (Cy5-GCC TCG CTG CCG TCG CCA (SEQ ID NO: 40)) and DNA4 (Cy3-TGG CGA CGG CAG CGA GGC-T₂₀(SEQ ID NO: 45)). The PcrA-RepD interaction assay involved preparation of the RepD-oriD DNA adduct as described in Slatter et al (2009) supra. A biotinylated oriD DNA substrate was constructed by annealing oligonucleotides DNA1 (CTA ATA GCC GGT TAA GTG GTA ATT TTT TTA CCA CCC AAA GCC TGA AGA GCT AAT CGT TCG G (SEQ ID NO: 46)) and DNA2 (biotin-CCG AAC GAT TAG CTC TTC AGG CTT TGG GTG GTA AAA: AA TTA CCA CTT T₁₅ (SEQ ID NO: 47)). In one chamber, only oriD DNA (50-100 pM) was immobilized on the surface. In a second chamber the RepD-oriD DNA adduct was immobilized. 100-500 pM dual labeled PcrA-DM1 was injected into the chambers in buffer B (10 mM Tris [pH7.5], 10% glycerol, 15 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 3.4 mM Trolox, 1% (v/v) gloxy, 0.2% (w/v) glucose). Short movies of multiple chamber regions were recorded. Since the two Cys residues of PcrA-DM1 were randomly labeled with Cy3-Cy5 mixture, each movie contained a brief initial 633-nm laser excitation period to determine the molecules with a fluorescent Cy5, followed by turning on the 532-nm laser for Cy3 excitation. Only the PcrA-DM1 molecules with a colocalized donor-acceptor pair were factored in the E_(FRET) histograms.

smFRET signals were acquired by an Andor EMCCD camera operated with a custom software at 16-100-ms time resolution. E_(FRET) was calculated as described in R. Roy, S. Hohng, T. Ha, A practical guide to single-molecule FRET. Nat Methods 5, 507-516 (2008). Unwinding periods were measured as described in the text. The fraction of unwinding events was calculated as the proportion of the all DNA binding events that displayed an E_(FRET) increase phase. Error bars were calculated according to Clopper-Pearson binomial proportion confidence interval method (C. J. Clopper, E. S. Pearson, The use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika 26, 404-413 (1934)).

Example 6 Optical Tweezers Assay

The optical trap handle was a 6098-bp long DNA, amplified from λ-phage DNA and flanked by a 5′-biotin and a 3′-(dT)_(10,15,75) overhang (SEQ ID NOS 33-35, respectively) on the other end. First, a 5′-tailed 6083-bp fragment was amplified by the auto-sticky PCR reaction (J. Gal, R. Schnell, S. Szekeres, M. Kalman, Directional cloning of native PCR products with preformed sticky ends (autosticky PCR). Mol Gen. Genet. 260, 569-573 (1999)) using primers P1 (biotin-GGC AGG GAT ATT CTG GCA (SEQ ID NO: 48)) and P2 (GAT CAG TGG ACA GA-abasic-A AGC CTG AAG AGC TAA TCG TTC GG (SEQ ID NO: 49)). Subsequently the amplicon was annealed and ligated with oligonucleotide DNA5 (TTC TGT CCA CTG ATC-(T)_(10,15,75) (SEQ ID NOS 50-52, respectively)) to create the 3′-overhang for the initial helicase binding (10, 15 or 75-nt, as specified in figures). DNA beads were prepared by adding biotinylated 6-kbp DNA to the streptavidin-coated polystyrene beads (0.79 μm in diameter, Spherotech, Lake Forest, Ill.), and incubated at 25° C. for 30 min. Protein samples were pre-incubated with biotinylated anti penta-histag (SEQ ID NO: 44) antibody (Qiagen, Valencia, Calif.) on ice for 1 hour. One microliter of this mixture, 1 μl of streptavidin beads, and 8 μl buffer (100 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10% glycerol (v/v)) were mixed and incubated for 30 min on ice to make the protein coated beads. Reactions were performed in laminar flow chambers that were designed and assembled as described in Z. Qi, R A. Pugh, M. Spies, Y. R. Chemla, Sequence-dependent base pair stepping dynamics in XPD helicase unwinding. Elife (Cambridge) 2, e00334 (2013). Reaction buffer C consisted of 100 mM Tris pH 8.0, 15 mM NaCl, 10% (v/v) glycerol, 10 mM MgCl₂, and an oxygen scavenging system (100 μg/ml glucose oxidase, 20 μg/ml catalase, and 4 mg/ml glucose) to reduce photo damage to the sample (M. P. Landry, P. M. McCall, Z. Qi, Y. R. Chemla, Characterization of photoactivated singlet oxygen damage in single-molecule optical trap experiments. Biophysical journal 97, 2128-2136 (2009)). The reaction chamber contained two laminar streams of buffer C with different ATP, ATP-γS and SSB concentrations as described in the text. The dual-trap optical tweezers were set up and calibrated as described in (C. Bustamante, Y. R. Chemla, J. R. Moffitt, High-resolution dual-trap optical tweezers with differential detection. Single-molecule techniques: a laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2008); K. Berg-Sørensen, H. Flyvbjerg, Power spectrum analysis for optical tweezers. Review of Scientific Instruments 75, 594-612 (2004)). All measurements were recorded at 100 Hz with a custom LabView software (8.2; National instruments, Austin, Tex.) and smoothed with a 100 Hz boxcar filter. In the “force-feedback” mode, unwinding was allowed to occur against a constant force of 10-22 pN (as specified). The contour length of DNA was calculated from the measured force and end-to-end extension of the molecule and using the worm-like chain model (persistence length of 53 nm, stretch modulus of 1,200 pN and distance per base-pair of 0.34 nm). The velocity of DNA unwinding in the force feedback mode was determined from a linear fit of the contour length of DNA in a sliding window of 0.2 s (21 data points). Pauses longer than 0.2 s were removed and then the velocity was averaged in 1 s bins. Error for the fraction of unwinding events per tether formation was calculated with the Clopper-Pearson binomial proportion confidence interval method (Clopper et al. (1934) supra)).

The force dependence of Rep-X unwinding activity was measured in the “fixed-trap” mode, by stopping the force feedback. The force data (100 Hz) was smoothed with a gaussian filter (by applying a 33-Hz moving average filter 10 times). Paused regions (velocity <10 bp/s) were removed. The pause-free unwinding velocities were calculated and normalized by the velocity at 20 pN for each molecule, and binned against the dynamic force values up to 60 pN to create the V_(norm) vs. F plot (FIG. 3F). We previously found that the force response of our trap was linear against bead displacements up to 72 nm (determined in a separate experiment measuring where the force vs. extension curve of dsDNA started to deviate from the theoretical worm like chain. At a trap stiffness of 0.167 pN/nm, the deviation occurred above 12 pN). Hence we calculated the maximum reliable force to be at least 59 pN at a trap stiffness of 0.82 pN/nm.

Example 7 Ensuring Monomeric Rep-X Activity in Optical Tweezers Assay

We considered the possibility that the highly processive unwinding observed in our optical tweezers assay was caused by multiple Rep-X acting on the same DNA. If multimeric Rep-X had been required for highly processive unwinding, then the majority of binding events (i.e. formation of a tether) would not have displayed unwinding activity, because single Rep-X binding is the statistically the most probable event during the brief period of contact between the two beads. However, the majority of tethers formed displayed highly processive unwinding, suggesting that the processive unwinding is caused by a single Rep-X protein.

To further establish that the unwinding of the 6-kbp DNA was achieved by single Rep-X molecule, we repeated the experiment using beads incubated in lower concentrations of Rep-X, thus decreasing the number of Rep-X molecules per bead. Consequently, Rep-X binding (tether formation) took longer and required more trials of bumping the two beads. As the Rep-X concentration was lowered. (20 nM, 4 nM and 0.4 nM) during the pre-incubation with 20 nM biotinylated antibody, the efficiency of tether formation was also reduced (7 out of 11, 9 out of 27 and 2 out of 16 beads, respectively). However, the subsequent unwinding was still the prevalent behavior (7 out of 7, 8 out of 9 and 2 out of 2 tethers, respectively).

As another test to ensure that the highly processive unwinding was due to a single Rep-X molecule, not multiple molecules, we compared the unwinding reaction of DNA with 75nt vs. 10- and 15-nt 3′ overhangs. Since the footprint of Rep is reported to be 8-10 nt (S. Korolev, J. Hsieh, G. H. Gauss, T. M. Lohman, G. Waksman, Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell 90, 635-647 (1997)), 10 or 15-nt overhang would increase the chance of single Rep-X binding. Rep-X exhibited the same highly processive behavior on the short overhang DNA molecules (17 out of 18 tethers formed with 10- and 15-nt overhang DNA vs. 21 out of 22 tethers formed with 75nt overhang DNA, FIG. 3B, C), further indicating that the high processivity of unwinding is the property of a Rep-X monomer.

To test the possibility that the unwound ssDNA interacted with additional Rep-X on the bead surface, possibly increasing the processivity of unwinding, we added 66 nM of E. coli ssDNA binding protein (SSB) in the unwinding reaction stream in order to render the unwound ssDNA inaccessible to other Rep-X molecules. Inclusion of SSB did not change the highly processive behavior of unwinding (17 out of 18 tethers formed in the absence of SSB vs. 21 out of 22 tethers formed in the presence of SSB, FIG. 3B), suggesting that DNA unwinding by Rep-X is highly processive whether the unwound ssDNA is sequestered by SSB or not. This observation is probably due to the design of the dual optical tweezers assay, in which the DNA is under tension only between the “front runner” Rep-X molecule and the streptavidin on the other bead. Binding of a second. Rep-X to the already unwound ssDNA should not affect the measurements because the second Rep-X, which is also tethered to the bead, cannot interact with the front runner that is tethered elsewhere on the bead.

Example 8 Selection of Crosslinking Sites and Crosslinker Length

Open (inactive) and closed (active) form crystal structures of Rep and similar helicases were used as a visual guide. The target residue pair for crosslinking and the crosslinker were selected based on these criteria.

One target residue of the target residue pair should be located on the mobile 2B domain and the other target residue should be located on the immobile body of the helicase (for example on 1B or 1A domains). Preferably, target residue pair should not be part of functional helicase motifs known in the literature to prevent detrimental effects of amino acid engineering. Preferably the target residue pair should not be conserved residues. Preferably the target residue pair should be as far away as possible from the ssDNA binding sites. These measures reduce the potentially detrimental effects of the target residue mutations and crosslinking on the basic translocation function of the helicase.

The target residues should be as close as possible to each other in the closed (active) conformation of 2B domain, and at the same time should be as far as possible from each other in the open (inactive) conformation. For example, the distance between the target residue pair should be less than 15 A in the closed form (measured from alpha carbon coordinates) and should increase by more than 30 Å during transition to open form, so that a short crosslinker can prohibit the transition to an inactive (open) form. Residues that satisfy such criteria can be determined for helicases with known crystal structures in closed or open forms.

By sequence alignment, the corresponding crosslinking target residues can be found in helicases with unknown structures to convert those to superhelicases, as well. Sequence homology models can also be employed.

Target residues should be preferably on the surface of the protein, and their side chains should be facing outward and more preferably facing toward each other.

The crosslinker should be as short as possible, preferably only long enough to efficiently link the target residue pair in the desired conformation. Crosslinker length should be considerably shorter than the distance between the target residues in the unwanted conformation.

A representative 56 Rep homologs/orthologs with 90% identity to and 80% overlap are shown in Table 4, which are also shown in FIGS. 9A-G. The target residues of FIGS. 9A-G were selected from one residue from domain 1A or domain 1B, and one residue from domain 2B which satisfy the all these considerations. For PcrA, or a homolog thereof, the target residues are selected from residues 92-116 of domain 1A or 178-196 of domain 1B, and 397-411, 431-444 or 526-540 of domain 2B. For Rep, or a homolog thereof, the target residues are selected from 84-108 of domain 1A or 169-187 of domain 1B, and 388-402, 422-435 or 519-536 of domain 2B. For UvrD, or a homolog thereof, the target residues are selected from residues 90-114 of domain 1A or 175-193 of domain 1B, and 393-407, 427-440 or 523-540 of domain 2B.

TABLE 4 Rep homolog Organism REP_BUCAP Buchnera aphidicola subsp. Schizaphis graminum (strain Sg) REP_BUCAI Buchnera aphidicola subsp. Acyrthosiphon pisum (strain APS) (Acyrthosiphon pisum symbiotic bacterium) REP_ECOLI Escherichia coli (strain K12) REP_HAEIN Haemophilus influenzae (strain ATCC 51907/ DSM 11121/KW20/Rd) REP_SALTY Salmonella typhimurium (strain LT2/ SGSC1412/ATCC 700720) A0A077ZIR6_TRITR Trichuris trichiura (Whipworm) (Trichocephalus trichiurus) S3IEG5_9ENTR Cedecea davisae DSM 4568 J1R585_9ENTR Kosakonia radicincitans DSM 16656 K8ABZ8_9ENTR Cronobacter muytjensii 530 A0A060VJ91_KLEPN Klebsiella pneumoniae A0A090V5M6_ESCVU Escherichia vulneris NBRC 102420 A0A083YZC2_CITAM Citrobacter amalonaticus A0A0J6D7T8_SALDE Salmonella derby A0A085ITL8_RAOPL Raoultella planticola ATCC 33531 E7T4Q1_SHIBO Shigella boydii ATCC 9905 A0A085GMM2_9ENTR Buttiauxella agrestis ATCC 33320 A0A085HAK1_9ENTR Leclercia adecarboxylata ATCC 23216 = NBRC 102595 D4BE16_9ENTR Citrobacter youngae ATCC 29220 A0A0H5PMJ7_SALSE Salmonella senftenberg A0A0J1JQT3_CITFR Citrobacter freundii A0A0J8VI05_9ENTR Cronobacter sp. DJ34 F5S3F4_9ENTR Enterobacter hormaechei ATCC 49162 D2ZMA5_9ENTR Enterobacter cancerogenus ATCC 35316 A0A084ZTW9_9ENTR Trabulsiella guamensis ATCC 49490 A0A038CLT3_RAOOR Raoultella ornithinolytica (Klebsiella ornithinolytica) Q8Z385_SALTI Salmonella typhi Q83IX8_SHIFL Shigella flexneri A0A0D5WYP4_9ENTR Klebsiella michiganensis A0A0H3FM31_ENTAK Enterobacter aerogenes (strain ATCC 13048/ DSM 30053/JCM 1235/KCTC 2190/ NBRC 13534/NCIMB 10102/NCTC 10006) (Aerobacter aerogenes) A0A0H2WUK6_SALPA Salmonella paratyphi A (strain ATCC 9150/ SARB42) A0A0H3H1F3_KLEOK Klebsiella oxytoca (strain ATCC 8724/DSM 4798/JCM 20051/NBRC 3318/NRRL B- 199/KCTC 1686) X7I146_CITFR Citrobacter freundii UCI 31 A0A0H3CTF5_ENTCC Enterobacter cloacae subsp. cloacae (strain ATCC 13047/DSM 30054/NBRC 13535/ NCDC 279-56) D2TH67_CITRI Citrobacter rodentium (strain ICC168) (Citrobacter freundii biotype 4280) Q329V6_SHIDS Shigella dysenteriae serotype 1 (strain Sd197) W6J7C4_9ENTR Kosakonia sacchari SP1 I2BE87_SHIBC Shimwellia blattae (strain ATCC 29907/ DSM 4481/JCM 1650/NBRC 105725/ CDC 9005-74) (Escherichia blattae) B5EZ38_SALA4 Salmonella agona (strain SL483) A0A0F5SGU2_CITAM Citrobacter amalonaticus G9YY11_9ENTR Yokenella regensburgei ATCC 43003 A0A090UXU3_9ENTR Citrobacter werkmanii NBRC 105721 A9MJ31_SALAR Salmonella arizonae (strain ATCC BAA-731/ CDC346-86/RSK2980) Q3YVI6_SHISS Shigella sonnei (strain Ss046) D3RHB6_KLEVT Klebsiella variicola (strain At-22) Q57HT8_SALCH Salmonella choleraesuis (strain SC-B67) B5RFS5_SALG2 Salmonella gallinarum (strain 287/91/NCTC 13346) A0A089Q204_9ENTR Cedecea neteri A0A0H3BNR1_SALNS Salmonella newport (strain SL254) C9Y4T0_SICTZ Siccibacter turicensis (strain DSM 18703/ LMG 23827/z3032) (Cronobacter turicensis) B7LU77_ESCF3 Escherichia fergusonii (strain ATCC 35469/ DSM 13698/CDC 0568-73) A0A0H3TAW8_SALEN Salmonella enteritidis G2S5J6_ENTAL Enterobacter asburiae (strain LF7a) A0A0F7JC30_SALET Salmonella enterica I A7MQI4_CROS8 Cronobacter sakazakii (strain ATCC BAA- 894) (Enterobacter sakazakii) L0M8J0_ENTBF Enterobacteriaceae bacterium (strain FGI 57) A0A0K0HFU2_SALBC Salmonella bongori (strain ATCC 43975/ DSM 13772/NCTC 12419) A8ACT1_CITK8 Citrobacter koseri (strain ATCC BAA-895/ CDC 4225-83/SGSC4696)

Use of shorter crosslinkers increase the efficiency of crosslinking reaction by favoring the intramolecularly crosslinked species rather than intermolecularly crosslinked multimeric species. These rules also ensure that the 2B domain is restricted to the active (closed) conformation, and cannot attain an open (inactive) conformation. Thus conformational control is achieved, and the possibility of 2B domain to swinging open to access an inactive (open) conformation is virtually eliminated.

Without being bound by theory, one possible explanation for the super activation would be the decreased dissociation rate due to the crosslinked protein encircling the ssDNA strand (indicated by the crystal structure, so that the protein cannot dissociate from the ssDNA easily. However, it was found that despite both Rep-X and Rep-Y encircling the ssDNA (as indicated by the crystal structure), only Rep-X was super-active. Thus, in order to create the super active helicase, immobilization of the correct conformational state of the 2B domain is necessary.

Example 9 Identifying Suitable Crosslinking Sites in Homologous Helicases

Based on the crosslinking target site selection criteria established in Example 8, potential crosslinking target residues in helicases were determined using known crystal structures. By sequence alignment and structural homology modeling, the corresponding crosslinking target residues are identified in helicases with unknown structures. Subsequently these helicases can be converted to superhelicase forms. For example, based on the criteria that the distance between the target residue pairs should be less than 15 Å in closed form and should increase by more than 30 Å in open form, we identified the residues in Rep, PcrA and UvrD helicases as shown in FIGS. 9A-G. Homologous helicases are identified, for example, by 50% sequence identity and 80% overlap to the helicase with the known structure. For example, we found 3147 such proteins homologous to E. coli Rep, 1747 proteins homologous to B. st PcrA, and 1209 proteins homologous to E. coli UvrD helicases were found (Tables 5-7, respectively). Then the corresponding crosslinking residues are identified in any of the homologs. For example, we chose an example of 56 Rep homologs (Table4), and found the regions where the crosslinking residues can be engineered (FIGS. 9A-G). Despite the fact that the three model superfamily 1 helicases, UvrD, Rep and PcrA, have only 35-40% sequence identity, they exhibit >90% structural homology according to their crystal structures. Hence it is reasonable to expect a highly similar structural homology from the proteins with 50% identity to and 80% overlap to the helicase with the known crystal structure; these are suitable candidates for crosslinking in the superhelicase (−X) form.

E. coli UvrD (ecUvrD) has 33% sequence identity with E. coli Rep (ecRep) and 42% sequence identity with Bacillus stearothermophillus PcrA (bsPcrA). Highlighted regions in FIGS. 9A and 9G show the crosslinking sites obtained from the open form and closed form crystal structures and the criteria established in Example 8. These regions align well in the sequence showing that a sequence alignment can be used in helicases with unknown structures to determine the crosslinking target sites in helicases with unknown structures. For example, the crosslinking regions (boxed sequences of FIG. 9G) in D. radiodurans UvrD (drUvrD) were found by aligning its sequence to bsPcrA, ecRep and ecUvrD, 1A/1B residues: 92-116, 182-200, 2B residues: 400-414, 434-447 and 528-544. drUvrD (Q9RTI9) has 33%, 36% and 41% sequence identity to bsPcrA, ecUvrD and ecRep, respectively. These four proteins have 21% sequence identity as a group. Only closed form crystal structures of drUvrD are known. Boxed regions shown in FIG. 9G are shown in the crystal structure of drUvrD (FIG. 11) to demonstrate the suitability of the regions for crosslinking.

D. radiodurans UvrD (drUvrD, Q9RTI9_DEIRA) has only 1 Cys residue, and a crystal structure is known. drUvrD has 31 entries in the 50% identity cluster of the Uniprot database, some of which are mildly thermophilic (40° C.-68° C.; optimum growth at 60° C.), making them better candidates for helicase dependent nucleic acid amplifications. In certain exemplary embodiments, a suitable UvrD helicase is selected from following species: Deinococcus geothermalis, Meiothermus sp., Marinithermus hydrothermalis, Marinithermus hydrothermalis, Oceanithermus profundus. Selected thermophilic ortholog species of drUvrD are shown in Table 8.

In another embodiment, the helicase is selected from those shown in Tables 9 and Table 10.

TABLE 5 List of 3137 unique non-redundant helicases that have 50% sequence identity and 80% overlap with E. coli Rep. (Uniref50_P09980 cluster, citable UniProtKB and UniParc accession numbers are shown). P09980 UPI00051877AD UPI00050997D4 A0A063KTD1 W0QF97 A0A0C3I5L6 A0A0G3HMG0 UPI0002CB7C3E UPI00041BBC9F A0A0F9UW26 A3MZ01 UPI0005EDEB6E A0A069YUU2 UPI0002CB6CB4 Q31J65 UPI00057A0DA3 A0A0A7MFM3 Q4F7B3 A0A069XK09 UPI0003EF7150 UPI0004A7511F UPI00042735E9 UPI00037127A9 H2IB31 A0A090J554 A0A0B5IRM0 J3VUI0 A0A0B2JU34 W0QB54 UPI00039DFE76 J2LMP0 UPI0005F8A649 B3PF82 A0Y242 D9P8T6 UPI0002D3CD90 A0A0E1CLG8 UPI00036A6E72 W1J4L6 G7F259 B0BT27 UPI00031FC355 W8VIF4 E7T4Q1 A0A068QMH5 F3BJI5 E0EII5 A0A034TPW5 A0A0J2JAV4 UPI0002C94EDF UPI000645DEF6 UPI0004641B09 A0A011P892 UPI0003B1A99F A0A0H4Z590 UPI0004A19D90 A0A0J1C9T2 W1Z619 UPI00047C9D5E A0A090P8C9 A0A0H4YMK4 A0A024KL85 A0A077P2A7 N6W1D7 W0QMN7 U3AJD4 A0A0H4ZZ54 A0A029K3Y0 A0A077PGN3 G7EU46 B3H0C1 A0A0H0Y6H0 W1HB14 A0A029LST3 A0A077Q5V6 UPI0003F6330E E0FKS8 D0WV97 A0A0H4ZHU7 A0A074IDU1 A0A0B6XFA1 UPI0004175A32 E0E690 UPI00039CA46E A0A0G3T252 A0A070UMT6 D3VHW6 G7EBR9 E8KIM6 K5UKZ2 A0A0H3GKB6 A0A0H0NZ89 N1NN52 UPI0002317DDE KOFXU5 A0A061Q0T1 A0A0K0GSG9 UPI0004DACF34 A0A0A8NWA1 UPI000412695D A0A0D6UGN8 D0X594 A0A0G8G0X7 UPI000543FDCF W1J8H9 A0A099L6T2 M9X512 UPI0006814400 A0A060VJ91 A0A0H3MJI3 A0A077NN58 UPI0002DA5F7A D9P4Y2 UPI0006AA085C W1HUU8 UPI0002A2C4E1 A0A068RA85 UPI0003162143 UPI0002556C8C UPI00039C63E0 W1EG06 A0A0H0KMW7 A0A0A8LWM3 U1MBQ7 A0A0B0HCL6 UPI0005EFDC1A W1HYL7 F4NQI1 A0A077PLN5 U1JK11 UPI00041A33FA UPI00063C4F58 W1EC12 A0A0G9FGL3 A0A077PEC1 U1IZP2 A0A081FYF7 UPI0002B70576 V0AP50 UPI0005305422 D3V6L9 Q3IJF4 A0A0B3BWV0 UPI00023755B4 W1E3B2 I2X0N8 A0A0J5FTN5 E6RJ61 A0A0B2DBM6 UPI00069FBA0A A0A080SZ33 UPI00063C108B A0A077N2X7 Z9K3Y4 UPI000337D905 A7K6B6 A6TGG4 UPI00053AB8E4 UPI00037EA902 G7G5E8 D2TCP1 U4DZN2 W1BG54 UPI000390341B A0A094NXP4 UPI00034C744C UPI0001960924 L8XB50 Q57HT8 UPI0002C97E36 F9QBX8 A0A0F4PWX2 V5Z3P5 UPI0005975B9F C0Q2V7 T8JFG3 UPI000364DEC7 I3CJI7 UPI00065FAA51 UPI00039C5A41 A0A0G2NT58 A0A0J0IRL7 A3V094 A1T091 UPI00054F6EBB UPI0005F0AEC8 V2QUW5 J1GHC8 UPI0002D3C2F3 UPI00036133E4 UPI000554232A A7N1B5 B5RFS5 UPI0005EFAD7E F9SI11 A0A0A8UTG2 UPI00044B0A82 U4K9S4 H3N5E4 A0A0D7LCG7 UPI00065FA69F UPI0002624F9F W0T2G5 U3ATU8 UPI00030AD7A3 UPI0005EFAEC1 UPI0003122A61 UPI000465C470 A0A014LYL5 A0A0C1YTU5 A0A0J2K868 UPI00058EBC4A UPI00030ED944 UPI0004E14814 E3DCZ6 A6AVT6 A0A0E2RBB6 A0A0J5E886 UPI0003151B6B A3YC72 UPI00069D7BD3 UPI0003B1A0B5 A0A0G3S4X6 UPI0004D3B9EA UPI0002EE9ECE A0A0F4QZY4 D8MKR6 UPI0006A5B784 A0A0H3H1F3 UPI0005B485C4 F6CZU1 UPI00036B36CA UPI00058F6E3E A0A0D0JDJ7 A0A068HB64 V2SM85 A6W1H3 B5JVS4 I3IF96 UPI00069EC01E A0A0J8Z8W0 UPI0003AC5FE5 X7E960 M4U1U4 UPI00066FB81F UPI0006A5FC2D UPI00066AC9B7 UPI00057BEBAB W1RWL7 A0A0E4C1E5 UPI0005866DF0 UPI00026C4ED4 H3MDG6 A0A0C2ELY7 UPI00037950B0 C4K8X2 UPI00037B74A6 UPI0005EF44E2 UPI0002F9F873 U9Y0B8 A0A0J8GYA3 UPI0004762303 V5VG28 UPI0005EFA2F9 A0A0A0FJZ3 A0A0H0JVJ4 UPI0003F7784F UPI000558F88A D0C9R6 A0A0C1Z376 A0A077ZIR6 UPI0002CC3FC4 I1E2U9 A0A0E9M3L1 A0A014AME8 A0A061PWI7 A0A0H7U3H0 A0A0D1Q3M4 Q0VLG2 UPI000415F58C A0A062IS38 A0A090RFC6 A0A0H7UZL4 N2JD25 B4X088 UPI0003736C3A A0A062RV86 F7S1D5 W1YGM8 N3FAJ0 A0A095S578 UPI00047DD116 A0A014QUN9 UPI00037B6B31 W1F515 D7XDE9 U7FXU8 UPI00051B59BB A0A0J1AD48 UPI0004DB80FB W1AVB8 J1R585 L0W9M5 A3WIV3 A0A011J490 UPI0002559C1C UPI00050B4286 A0A063XPH7 A0A095SJP8 UPI00058ED431 A0A009L4W9 UPI0004226D1B UPI000699BCA0 UPI00046E61E1 UPI0003096010 A0A0B7IYA3 A0A062ET27 UPI0005A04CC3 UPI0001BCF307 UPI0005CFB922 Q5QZ79 A0A0D7FE58 A0A062B4C4 UPI00036751C4 UPI000291E920 F1ZPM5 A0A0B4XU81 N2J9F8 K6NBZ9 UPI0004A71315 UPI0005CECD30 E9Z170 V4LHL4 H0JBY0 A0A062HCL9 G7LUC7 UPI000699485E I2RDB4 UPI00048D495E UPI000307EC21 U1UEQ4 C6DHF7 UPI0006995E89 G9YY11 UPI00066EC8FC UPI00056B6FFA N9HSF4 A0A0G4JUN2 A0A0H7XVC5 UPI0005A8BE84 A0A0E0Y6R4 UPI000694928A A0A009GJZ6 UPI0003AAB78C A0A0H7ZBU6 B7N271 F2G1X5 W9SXN9 A0A009HQM2 UPI0003A1DD75 A0A0H8QAW7 A0A029IHD3 S5C3L8 G2E0G6 L9NZJ0 UPI0003AA3036 T9GN10 A0A029HDM3 S5AI16 H5V6K1 A0A011IKX6 UPI00039E8574 U9YW53 UPI00005EFD38 UPI00057CAFFD K8AD99 A0A062FYV6 UPI0003A8ECA5 E1ITJ2 UPI0005175495 K0CYB3 UPI0002F4EC6B N8ZCZ6 UPI0003A98BB4 A0A029L915 UPI0004D67711 K0EB21 K8AIR9 A0A022JJ04 UPI00039EFED1 S0UVF6 UPI00038F4A0A A0A0B3Y7T5 UPI0002F0AB46 K1FLW5 UPI000532BCE2 I2SR57 UPI0004ED858A A0A075P1K0 UPI0003747342 N8UCZ4 UPI0003AAAD9F I4S298 S1FND5 UPI000509BB4B A0A0D1MQD1 A0A010EBV1 U6ZJ94 D7ZUA2 S1LRE3 F5Z5Y6 F8FZI5 A0A009SCE5 UP10005539481 A0A069X2C0 S1CH47 UPI0003556595 UPI0004706F0C N9GH50 UPI00039F0A65 F3WQ14 L2VMG7 A0A010PKS2 UPI0004870A8C A0A0J1AU91 UPI000399CBB8 E1J5T7 UPI0005EDE9CE A0A0C5ITU1 UPI0005B76FDE A0A009JME7 UPI00039E8082 M9FM00 UPI0002CA0423 UPI0005D7D9DE UPI0004838E3B A0A062TQ97 UPI00039A51A2 S1HS46 UPI0004E384CD J2S9U8 A0A0C5RPA5 A0A009QEV1 UPI00039E8236 D7YBV4 UPI000675EB35 UPI0002E4097E F0E436 A0A010UBN4 V3TUV7 D7YG19 V0Z8Q3 UPI000641D99E J3E3S1 A0A062FMG3 A0A0A8FH34 V0RR51 D7ZDM9 J3D7I1 T2HEW7 A0A013G992 UPI000532D788 V0AF21 V5B2V3 J2T1B6 B0KQ68 A0A0J1A474 D2BYS8 E9YLU8 V1I8U1 J3EAI0 UPI00048196DC N8YU24 C6CGH3 I6CD40 T9SAP6 A0A0J6H1C3 A0A084C8X6 K6LHM0 A0A089V9A1 E1HWT5 A0A0I1J330 J2MEX6 A0A099E6T7 A0A062DHL7 A0A0A3ZID8 A0A070FCA9 A0A0J3J6C1 UPI0004653130 A0A088NVW6 A0A009TH79 Q9K599 V0TJ44 L0M8J0 UPI000370037D A0A0F7Y5R7 A0A0J0ZSY5 A0A0B2TLM6 S1H2A1 UPI0002C957E6 UPI00054B503C A0A099N7M2 F5I0P2 E0SKR5 E6BNJ7 UPI00033C37F9 UPI0005A8A319 V7DDK2 A0A010K6Q8 J8TIB8 D6I334 UPI0003EF8387 L1M697 L0FS45 F5IED5 UPI00050455DF S1ESS4 UPI00040AF69D A0A0F4VBH8 V9V750 N9KF69 A0A0B3XKC6 S1CGG8 W8XM69 J2XVS9 A0A059V3S3 S3TL87 D0KC43 I2WCN5 F0JWU2 J2NB92 B1J4P9 A0A009MSS6 A0A0H3IEB9 A0A070DIV3 A0A0J2D6H5 S6JBD8 UPI000627A5A1 A0A0J0ZPS4 A0A0J5XQ16 A0A079D8A1 L5GW15 J2UYW0 UPI0003A45A0C A0A010GGU7 UPI000505D45B A0A080I8Z6 S1P4F9 I4JZZ1 UP10004901875 A0A0J0ZKR3 A0A0J6AFU6 L4IUS8 UPI0006187646 A0A0J8G475 UPI000312CC5F B0V8Y9 A0A0B3Y0I6 L3IPS4 UPI0002CA3627 A0A038GJS8 V6JD09 A0A0E1PVY4 UPI0004E7AD0D V6FPL2 S1IH44 J2NTP4 A0A0C1IE71 A0A0E0WM10 UPI0005010995 F4T625 UPI0001FB5104 UPI00054BC40B U5VL93 A3M323 A0A0E2ZQ64 A0A073VUA5 UPI0006A60648 A0A0J6JAQ4 UPI0003B82996 A0A0J0Z9E3 A0A0J5UIH7 F4V8A1 UPI000666D818 A0A0J6I536 A0A089YA93 G2JCT3 A0A093TZI5 K3KG62 UPI00028CDADE K0WMX7 N9W558 A0A028IQG4 A0A0B2TAM5 M9E9G4 UPI0005182B07 UPI00069ECA48 V9X4R6 A0A0C0CBD9 A0A086ER15 D6JH94 UPI000246F098 UPI0003D2AA53 UPI0006836835 A0A0C0CIL5 J7L4K8 E0J414 UPI0002CBC547 A0A0C5ETI6 S2KQL9 UPI0003DF9CA6 A0A0B2W727 W8U2K7 A0A0A5IKM0 A0A075PD74 R9VCP0 A0A062DT29 A0A093S787 E8Y8U4 UPI00046F99E1 D7HTE0 U2U6Q9 N8VAV1 Q6CZD7 A0A0E0U640 UPI00066D2906 S6JE83 UPI000480BE0F A0A0F3L889 A0A0J5R7U1 A0A0E1SVC5 A0A0G3QEH2 J2LR66 UPI00062A24F9 V2VX37 A0A094RXX6 A0A0E2TLG7 W0AUJ4 J2Q4M5 UPI00040A96BC V2TSL6 UPI00057CA70B C6EG35 UPI0006A5EC48 UPI0003FAF552 A0A0J8UZS6 L9MEI1 UPI00057CDC60 Q3YVI6 R8WQW7 UPI000289EE48 I7BAY2 A0A009ZWD9 UPI0001A44592 J2YVU0 S0XDU9 UPI0006419BD9 A0A0A7PUM7 N9SD17 UPI00057F7392 A7ZTX9 UPI0002514D84 UPI0006A612B6 I3UUX4 N9C3A1 UPI000652BF86 A0A0E1M3S0 UPI0005CF34C7 A0A0F7A011 A5WAY2 UPI0004497B0F UPI0002D49EE5 I6BAF2 UPI0005AA6C75 F3JF49 Q88CB7 UPI0002BB404B U1LCU7 A0A090NEE8 UPI0002CB348F L7H1R3 A0A0A7JV78 UPI0004457216 U1JRW9 A0A0A7A0X5 UPI00039014F0 Q500M3 UPI000417E5AB UPI00044C3A7B A0A0F4P9F7 E2X573 N2Q8R5 S3NDD8 Q1I2W2 A0A0A7XTN0 A0A0F4PG18 Q329V6 UPI0003B29D20 F3GZK5 UPI0004A76E61 A0A022ILU3 A0A0C1LH68 F4TMM3 UPI0005F81F2E UPI0005CA31AB A0A0E9ZS43 A0A066PI57 UPI00061D116D U9YTK0 UPI0002CA5EAC UPI00035D1EA1 UPI000615E3F7 A0A062G9S1 A0Y9C9 B3X5G0 UPI000462FEC5 W0MPM6 A0A0H5W4I1 L9LZ08 Q2S8F2 I6CYC2 A0A070DGP2 A0A0A1Z8J9 A0A0H6ING7 A0A010KUF7 K2JN56 A0A0A0GP02 S3IEG5 W2DE98 UPI00069F04C6 K9BCT1 UPI00067E742C F4SRI4 UPI0002CC411A A0A0F4TYK8 UPI00053BC90E A0A0E2G0N2 A0A084IL47 UPI0006981AFC UP10005718CCF UPI00041035FD UPI00069E6D92 A0A0E2GP36 IJPT0006697E7E V0ST83 UPI00036A6951 F2ZPZ1 A0A0H7D3K2 UPI00044F9626 C9PMC5 T8WTG6 UPI0002A38058 UPI00045100E9 D2YQS5 UPI00044ACF5B UPI000402FD5E A0A071C0Z5 A0A0I1VLP8 UPI0006458FD3 D2YET1 UPI00044D2485 UPI0004802687 V0XTH9 UPI0002CB6DE1 A0A098SWG7 A0A085TFB6 UPI0005AB250E H0PYR0 D8C4A4 T5NUB4 A0A0D1NXC0 UPI00051D019F UPI00044F9882 UPI00037C100B T9AU26 A0A0D7F5Y8 UPI000589F9C7 A0A0F2IJ11 A0A013NSE0 UPI00067D4203 E1HNU3 A0A070RX93 UPI000614591D UPI00050288CA UPI0003558246 UPI0002FE41B0 H4URG0 H3MUT6 UPI000370CF7F A0A099MUD3 A0A062L2N7 UPI0005BC9A68 A0A070SPZ6 A0A070H7B2 UPI00061DB54E UPI00042A59FC A0A014DQT7 A0A0J6NLY1 A0A073HDV7 UPI00025B202A UPI00048A25A1 C9QBF3 A0A014BGK8 A0A0J6KGG3 S1HND8 A0A064CY65 UPI000463D380 UPI00028D4D13 A0A014E4S6 UPI0005BBAD58 A0A079FCR6 UPI0004D5922D A0A0F4SXJ2 A0A085PFD0 UPI0002FB179F B0UTK9 A0A073UI24 UPI00038FE44C E2Y0S2 UPI0002F9A474 A0A0B2XRW1 Q0I2R4 V6FCI2 UPI00057C0F18 UPI00054B1EC3 UPI0005C96744 N8S5C9 A0A0F6RAV7 W1BJG8 UPI0004D7032E A0A0J6KIB8 A0A0H6I9T4 UPI0004537654 B7S2C9 A0A064T0J2 A0A0J4XAT3 UPI0004157785 A0A0H3AKZ5 UPI0004526A41 H1G3C3 A0A070Y1X0 UPI0002C8F3D6 UPI00031D1029 A0A0H3QD21 A0A014CAF7 UPI00048A6F57 A0A073GI88 UPI00061449A9 UPI00046A96D2 Q9KVH9 UPI00061AF1D4 K2KA81 V8KBQ0 A0A0F5SGU2 UPI000287BEB4 C3LQ06 UPI000665B4E7 UPI0005A75154 A0A070NWA9 A0A0C1ZV53 UP10004882B64 UPI00053C3831 N9IYA6 A0A0F6KJ15 A0A071AHD9 UPI0002CB0481 UPI0004937205 A0A0H6DK35 A0A062KZU0 UPI00053802DA A0A079GY22 A0A029P5M4 A0A0B8VQT6 A0A0H6WUV7 UPI0005A67365 A0A0E2LUW8 A0A074HJN3 UPI0002A43CB4 UPI0004E6F9D0 UPI0000F34C0E A0A013IDC6 K1JFB9 V1AR00 C9Y4T0 UPI0005B4FF04 A0A0H6U8U2 A0A013P0S9 A0A023RRT0 A0A080HY13 UPI00049ED8E4 K9NS95 UPI00069E3D50 A0A009LQZ8 UPI0005AA85F9 L3QA81 R5WQS0 J2QIN9 UPI00069F740E A0A062FFT3 UPI0005AA6DC7 I2X3U6 UPI0002C9A11F S6I847 UPI00069D81D6 UPI00056E426A UPI0005CE86ED L3K8G1 UPI0003901F07 A0A085URN7 UPI000472B301 A0A009EYA5 K1K4T1 A0A080GHT9 K8CHU4 UPI0006762E20 UPI0005B3F2F7 Q6FDR1 UPI0005C13448 A0A070PMD4 F5VKN6 A0A0D6C968 UPI000419FA2F A0A014CQR5 UPI00068176EC A0A079FM03 A0A0J1M6F0 A0A0D6BRV6 UPI00019F6F0B A0A014FSY6 UPI0005A8197B A0A071CG57 A0A0J1MPE2 UPI0004659D49 D7HG42 A0A014DDU2 UPI0005EE918B H1C5A6 K0XCY3 A0A0B7DG99 UPI0006154522 A0A062SQW5 UPI0005A7CCE7 A0A071DLR9 UPI000483904F UPI0005789654 UPI0004E46C62 A0A062MW11 A0A091AMQ6 V6E2G2 UPI0002CA7454 A0A099SK66 A0A0D1ESY4 F0KFL5 A0A0A5MTL6 D8AZU3 UPI0002CA7442 UPI0002887A69 UPI0004A3DD97 A0A010KWI7 A0A070ARJ5 I4J555 UPI0002C8C519 UPI0004E363B2 C2HS26 A0A009GU89 UPI0005B21AAA D8AJN2 UPI0002CAD3B5 UPI0004711D03 UPI0004939A0F A0A009RTE6 UPI0005B4D264 L3NTU4 UPI0005AB8E86 A0A0C2R6G2 UPI00041D5461 A0A013S7D9 UPI000206A54C A0A017JGH1 E3G401 UPI00036390E5 UPI00053C556B A0A011KJ92 UPI0005A9E387 E6B0V7 A0A0D6J066 Q4K3U4 UPI0006B26D10 W3T1R8 UPI00057A3319 G0F684 A0A0G3J3X1 UPI000467F1C8 UPI0003DDF141 A0A013T0B3 UPI00067160F8 T5ZL14 A0A0G2SEI1 UPI00035F9085 UPI00041C3DE6 A0A062GLJ2 UPI0005BE5278 A0A023LHN8 Q1R4G1 S6MAY1 D0IMK5 A0A022KT37 UPI0005B44CBF B1LLV1 A0A0H2Z4L9 A0A0D0PI96 UPI0004D45E01 A0A013LNZ0 A0A0A5LAX4 A0A0G3KG39 B7MGI8 W8PZL5 UPI00064AB4CA R8YFJ5 UPI0005A92C34 A0A0E3N453 A0A0E2KYV2 A0A0A1HZQ3 UPI00027344D7 A0A010JTN9 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A0A0H2VE70 A0A090SBH7 U4S459 M1FGB5 UPI000411120A V1L7R7 UPI0002CACCD9 G3IVH2 UPI0002CBFAC9 UPI000572A7F6 UPI0004221DBE A0A0J8M094 N2RLH9 UPI0004DF8545 UPI0003AC4DB6 UPI0004744F39 UPI000485D7F1 S4IGJ2 A0A0J1LII8 UPI00047F1C5C UPI0004ED825E A0A0F0DKQ4 UPI00045EA9DE S4JI21 UPI0003DE3024 UPI0001ECFED8 U4RRC2 V9HKH3 B9YZ61 V1PEP5 A0A0A5RMD0 P57654 A0A063CT47 C4L8M5 G2J035 A0A0J8MRJ4 A0A085ITL8 UPI0004B2EA54 U4RTG0 UPI000467E424 UPI0004844892 A0A0J8I253 A0A038CLT3 A9CX24 A0A0E1RML1 N6XTH8 L8D9A1 T2Q8L1 UPI000699D02C D4ZCT1 B8F834 N6X5A1 UPI0005DDA656 S4I960 UPI00053015A9 UPI0006860AC7 A0A084EXM6 S9ZLZ2 UPI0002DE6662 V7VTQ5 UPI0006A5F9C7 A0A080UY52 U4SEE3 N6YIH6 Q8D1K7 V1U623 UPI0002C9392A L7Z9H4 UPI00049FFDBE A0A0J8Q929 UPI000267BACB A0A0J8MLX1 A0A090V5M6 A8G826 UPI0004DFBD5A N6YW13 UPI0002679FF8 A0A0J8KIG6 UPI0005F02BB9 U2LS81 A0A0F5EXR4 C4ZMK0 UPI00036F034D A0A0J8LPE0 A0A0G2XKH3 UPI00020E94C4 A0A0F5EPE0 N6XY51 A0A014M0T5 A0A0J8JI45 I6FIC7 UPI0002DABF6B S6EML5 A0A0C3J2Q5 A0A0B6P3H3 A0A0J8NNW4 E7SHA3 L0MCV8 UPI000376F3D1 UPI00056F25D5 A0A0E8XKA8 A0A0J8M386 I6DJI8 A0A0G8B441 UPI0004921C45 E1SPI9 A0A0E1NWJ3 S4KQN0 B2TU14 UPI0002A42D68 UPI00036D6D23 UPI0003B58CFA A0A0H3B897 A0A0J8HDW7 E7TCP3 UPI0006652A04 UPI0006737FF4 Q65VJ0 A0A0H3QMK7 S4JDD8 Q31UK7 U2L789 A0A0F9VQX3 I3DJM6 Q66G21 A0A0J8M6F4 UPI0002C9AB91 UPI00061B5F40 A0A094MWI0 UPI0005CAE678 Q0WAE0 V2N1K8 R9VPF2 UPI000325C91D A0A0C1MVM0 UPI00069EE2BB A0A0H2YE35 S4K7P0 K8CWZ5 UPI0006688668 U1LW91 UPI00069E268B A0A0E8MJ70 A0A0J8LWR5 UPI0002C99AA5 A0A0J5CK09 A0A0F4QKC2 UPI00069D7318 A0A088KV63 A0A0J8HEC7 UPI00038FB95B S5EBF4 V4JJ09 UPI0003FDFDBC A0A0B6FKI9 A0A0J8JAK5 UPI0002C96518 A0A069CNT5 A0A0F6A4M8 A0A099KBQ4 A0A0H5LYI6 A0A0H5PMJ7 W9BJ58 A0A087L2V7 UPI0006270414 UPI0003FBA996 UPI0005DF0BEB V2NGI2 A0A0I2BS34 A0A086G6L5 UPI0006271440 UPI00048E7C37 A0A0H5H7D3 V1SGL7 W6J7C4 W0L9M1 UPI0003618E19 UPI000482708E UPI0006827E21 X5MWS2 UPI000500BF0E UPI00066770B0 UPI0004BB1652 UPI00051D9F62 A0A0H5JCZ5 T2PQT7 K8BQX4 D4E004 I3BQA3 A0A0E9BPQ7 A0A0H3NX30 A0A0J8KVX1 UPI0002CBEEC2 UPI0006615922 H5TCX1 UPI000495F3A1 UPI00067863B0 A0A021WMC9 A0A0J4V9J3 UPI000408D809 A0A0B7J8T9 D4GGK8 A0A0F6ZK05 A0A0J8J9B6 A0A0B7GDH2 A0A0F7HAB5 A0A0C5WV68 A0A0H3L1U7 A0A0H5ETP1 V1I8P9 Z5CRA3 A0A0G9N4S7 UPI0005CBA06D UPI00047515EA W0UFJ9 S4LIK5 B5XYZ5 A0A0F6KPT4 D0Z154 A0A0E9B357 UPI0005E9ED67 V2A692 D3RHB6 A0A0J8P0E6 A0A0D8MPB6 A0A0E9BHY8 UPI0005DD7BF8 A0A0J8L0F3 UPI0004D7628C S4YDU2 F2PBR6 UPI000468BFB9 UPI0005E79D67 V1MB85 UPI0003A3F66F A0A0J1YD25 A0A066RXJ4 A0A0A1B4E0 C4U266 B3YF36 A0A0J4SW69 A0A0J5CHL9 UPI000307D228 A0A0A3YME8 UPI0005DB455D S5ILC5 UPI000616F32D UPI00046870F9 UPI00040F27C9 U2MK51 UPI0003029766 A0A098GZB1 UPI0002CA634B A0A084A575 Q2C483 E0M1J4 A0A0H5IPV3 V1XST3 UPI00061449AA S0A849 A0A0D8SB06 A0A0F5XV05 A1JI64 V8ME39 UPI0006A97B14 UPI00048AA12D A0A0D8LLV6 UPI00036D7981 A0A0E1NPM1 V2JQQ7 UPI0005762BA1 UPI000487A67F Q1ZK16 UPI00034BF070 W8U8V7 A0A0H3S2U0 I6FYC6 D3HNV7 A0A0D8LUA2 UPI0004E1A398 UPI0005E99F5C X2KI94 A0A0J8VI05 C6C798 A0A0J1GP27 UPI000508FC34 UPI0005E00E96 A0A0H3SKK5 UPI000699B795 UPI0005615E14 A0A090RJP1 A0A0B1RCL6 A0A0B6HTA5 B5EZ38 C8TL39 UPI00040FFE96 A0A0J1H812 A0A0A6YFE9 UPI0005DDB975 A0A0H3ILF8 K3PM43 UPI0005684D6D UPI0005D2DDB6 UPI000534D94B UPI000173969B A0A023N7J2 A0A026HND2 UPI00037540FD UPI0005E822AD A0A0D8YE92 UPI0005E5EB23 V2P0B4 B1ERC7 A0A0J8YX51 UPI0005E9034E A0A059IDE0 A0A0E8MKS9 A0A0H3NHT9 S0TTI5 UPI0005E8F2FE A0A0D8QVE1 U1TNM8 A0A0H5NCP1 A0A0H4V1S8 UPI0005309AD3 Q605Y2 A0A0J1H2I7 E1SK66 A0A0B6HCA7 V7RZ28 K8BE03 UPI000308B214 A0A0B9GYU2 J3D9Z5 A0A0H5MV89 M7S3Q8 UPI00044CED1F UPI000413FA42 L8J7D2 UPI0002A6B5C9 A0A0H5PMI9 V1GIN0 K6KFS6 UPI00037FCD36 UPI0005959C45 U3TSP9 A0A0B6LTD8 S5HF97 UPI0006662A0A A0A068Z3I4 UPI000509EA27 A0A0F3LWH1 C4UBW2 V0GNY3 UPI000448B406 E9CNV6 N8QMA4 UPI0005C52466 UPI0005E9D7FF A0A0F0ITA3 UPI0004207793 A0A068RDD4 N9Q5H1 UPI00048B685F A0A0H5MA43 A0A0E1CUB8 UPI0004468690 UPI00039B0A85 S3TBN2 UPI00069BBC9C A0A0H4MX63 A0A0F7JC30 A0A0G3PTV9 A4BKH1 N9PM38 E6WHD8 UPI0005E84131 G4C8N2 A0A0J8ZB76 UPI0002E7F8E9 N9RHD1 UPI00050F5F1D C4T540 A0A0D5WN88 A0A0J9AH95 UPI00036F75F9 A0A009KDR6 A0A0F5FBT2 UPI0005EA618B A0A0F2ZS42 J5XNR1 UPI0004772558 N9MFP0 UPI00025848BE UPI0005E2712E V5ZRF3 A0A0E0WT30 D3SDE5 A0A022IP23 UPI0005358D4C A7FD44 V2D3X3 UPI00066507BD UPI000363C2C3 K9AX41 UPI00067631F8 C4SH32 B5Q5L3 UPI000665B1C4 UPI000381348D A0A009KTD8 UPI0005342DB0 UPI0005E88594 V7IN29 UPI000664F825 UPI00036A352B N8RGJ8 A0A0A3YQZ0 C4S2J8 E8XIM3 X4BC54 UPI0003638A7F V2TJN5 J2UWK2 UPI0005E15072 A0A0F6B977 UPI000517D3E6 UPI000368CFC7 V2UWT0 UPI00066187B2 UPI0005DBB3AC G5QS67 A0A0I0Y9Y8 UPI00036636AC N9QAE1 H3RIT7 W8G789 G5LVZ4 A0A070TA81 UPI0003689D3E N9KD21 D4HUI5 UPI0005E9DE69 UPI00067FCA22 F3VD48 UPI00035E83C6 N9TJB8 E5B0I1 UPI0005E6D02E G5PV49 H4V842 UPI00036EBDFE N9RBQ9 A0A0J8YTF5 A0A0B6GH85 G5RMY1 K8ABZ8 UPI00036CB07E N8XKN2 B2VG71 UPI0005E21E25 G5SIZ2 V5U423 UPI000371CE96 N9MQT1 UPI0002CAE332 C4ULT0 X3X6G6 A0A085HAK1 UPI00037E26A7 N9N9M9 A0A0A0ZCV1 A0A085U6D1 UPI0003063B27 H5A4A8 UPI0003719672 UPI000570AC25 UPI00048B3568 UPI0005E333BA G5PBC2 F5N8S8 UPI00056FCF90 UPI0005C62CED Q6LVY9 UPI0005E682AA G5QW51 A0A0J9AK28 UPI0003734BC2 N8VTA9 Q1YVV5 UPI0005E03B74 UPI0005E961AF UPI00067DF499 UPI0003714E69 N8VZT9 UPI00062F612F C4SRN3 G5MQG3 UPI0005CB2CD4 UPI000422ADAE N9TDU3 A0A034U1R0 UPI0005E51855 UPI0005F2CBD5 X4JC12 A0A0G3FYM1 N8V910 UPI00030424F2 UPI0005788705 W1XLV9 G5P130 UPI00035DF508 N8UMY4 U5A599 UPI00046D0E5D A0A0J0N6G9 UPI0003BCE0F3 UPI0003653619 N8U6B7 UPI0002F53240 M4R9M0 V2IST2 L0H0T7 UPI00035F46DD R9ATC8 UPI0003081AE5 UPI00046D66BE A0A0J8F6X7 UPI00062107C5 UPI00037A10E5 A0A0B0KKN3 UPI0002E72691 W0R997 V1SFT8 A0A0K0HP61 UPI00035F39C1 S7WVT1 B7VH95 A0A094IJP1 A0A0K0HFU2 A0A097QXR9 UPI000381010F N9KJH2 UPI0005F9EDF7 H8Z171 UPI0005AB2AE4 A0A0B8ZV63 UPI000368288B N9SJA9 UPI00063A1E73 C9R7F6 UPI00062081F9 UPI0005837FE4 UPI00036D136F N8W759 A0A0C1NZP7 E6KV30 A0A0J5KVY8 G9Y0Y6 UPI00037F35BB N8RKD0 UPI00064760CF UPI0005194C73 UPI00029C3FF8 A0A0C5VQS4 UPI00035EC95C V5C2I3 A0A0F4NTM0 U1S9B3 A0A0J1VMC9 W1FXH4 UPI000367CCE4 UPI00061832D7 A0A0A5HSY9 UPI000406D49B D2ZMA5 A0A085GZB9 UPI000423C045 A0A061JWL5 A0A0A6T9A9 UPI000660A603 UPI0003A1A991 UPI00062253DC UPI000373D30C UPI00036CBF04 UPI0005E2F450 G4A8W6 D7XM77 UPI0003A59E18 UPI00036243F4 W8QTS8 F9RNX1 G3Z8Y9 UPI0002677FD2 C9P8Z5 UPI00037D0EF9 A0A078LYT9 A0A0F4NNJ4 I1XRY3 D4BE16 F2JTM7 UPI00037114DA A0A099RR43 E8LUB9 UPI0006831C68 UPI0005794143 A4C7W3 B5FCU9 A0A0D9ATR1 UPI0002E710F1 G4AZ10 UPI000260B9F8 UPI00039F0D67 Q5E1T0 I4JQR3 UPI00031D8F80 L8UHA2 UPI00028307CF UPI0002558A91 B6EP51 I7A9J8 F7YIE8 UPI00067CF184 UPI0006660AB8 UPI0005B86EE4 UPI000247865A K5XG83 A0A066UM26 UPI0006815E34 A0A0E0VC55 A0A098G8R1 A0A090IP02 A0A0A1GK07 F9S363 H0KC96 T8Z104 UPI000326FBA0 T2L6T4 UPI00040F147B UPI0003102D21 A0A0E1YSI3 S0YAD1 UPI00037E68F8 L9UDC2 UPI0004194356 A0A099LPD8 X2JQA6 S0X3V2 UPI00034D7EBF UPI00037E36DB UPI0003B39C7B UPI0003167DE1 UPI00067FF093 S1DN83 UPI00056CF143 A0A0D7UZT0 UPI0005BA4855 UPI0002E0F676 C6AQX1 S0XCC9 A0A0B8V8Y9 A0A0F9VK34 UPI0005B7EB79 A0A0C2P7J1 UPI0006A71057 S1E4M9 U4TCI2 A0A0D5LWG2 A0A0C1EK43 A0A0C2JL07 G4ABR6 T9IPB3 A0A0H4R6J4 G4F9U9 U1AEW3 UPI00031E1DE0 L8UIY4 T6LBU7 A0A0B8USV8 A0A0B1PVT1 UPI000617AEB5 A0A0A5I590 Q7MYL0 S1GJ06 UPI000368EE93 H0J1C6 UPI0006182BB7 UPI0003043227 A0A022PH42 D8ADY5 A0A095VW14 UPI00048842D1 Q7NQR9 U3BS43 W3VA31 S0VUC6 A0A0C5UZZ5 UPI0002D5FF35 A0A0J6LGT4 UPI000571B2B4 A0A0A0CQ83 B7LU77 W8FU49 UPI000556BD4F A0A0D8ZDY8 A0A086WW56 UPI0006203273 A0A070K818 M5DYB2 A0A0C3I966 UPI0004907BDE F9T770 A0A0F7LMM1 A0A062XSU2 UPI00046D03A5 UPI0004843630 C5BIA4 UPI0005F11AED UPI00055C23C6 UPT0002C9B880 R4YVB5 F7SNI3 UPI0003800078 UPI000699D72A A0A0J9EYL2 UPI000651920D H2G1G7 UPI0004AB49AE UPI000382D783 UPI0005F118D8 C7BQK5 A0A0F3TFS9 UPI000379F9FB A0A060B1U5 UPI0004227F3C A5L7N9 U7R5K0 A0A0E2U8U7 UPI0001EC45E8 A0A0D6EF56 UPI00035C7304 UPI0001F55149 A0A081RWC7 C8TYS8 C6XCN5 G9EBD3 UPI000369273A U0FTU6 UPI00058BF6A9 A0A0J2E1P9 UPI00035FCCBC A0A0F4RA35 W8KPW4 UPI00030715AD T0PH03 UPI0005B2C8D4 UPI00058D9CB0 W1N5Q2 D5C0J9 UPI0006303856 A0A085JM95 UPI0002C95CB8 A0A0A0BIG5 UPI0004CE4C17 A0A0A3AKX9 E3BPB4 A0A095VZP6 A4WG32 UPI0002DE5ECC A0A081K8A4 UPI0006A9F0F0 E8MCD7 UPI00046F3C99 UPI0003420E0F F5T1S3 UPI000477FE94 F7NR22 Q7MQG8 UPI0004A3375F UPI00036F08F6 A0A0F9NIL3 A0A094JA28 A0A066T3V5 A0A087IWU5 A0A0F9VYU9 UPI0005C4EB5F C0N7C2 A0A090KED5 A0A080LJV3 UPI0006A98AC0 I1XM63 UPI0002C91779 F6DAI6 A6FE65 X2GZR9 UPI0006A98232 I2JF40 A0A078L9V2 UPI0005C9F562 UPI00030A12FE A0A0J5P3I3 UPI0002482DB8 F9ZY05 UPI000512B6F1 W0DYM2 UPI0002DF92C0 A0A0F2P6J6 F9REI7 A0A0F5V836 UPI0002CC209C UPI00022C089B UPI000464C875 W7R0N4 UPI0004F5E7D9 S6GDK2 A0A0D7LJA0 UPI0006844205 UPI000427CAB0 UPI0004752339 UPI00031835E8 S6HCZ4 A0A073VC48 W7QF72 B9CXM2 UPI0004E147DD UPI00031E4412 I8U5K5 X7I146 UPI00058DEE31 C5RZQ1 UPI000479BC17 A0A0H2MLA0 H3ZE84 X7HFY3 UPI000289826B UPI00035CAB1A UPI00047B5018 UPI0006195856 J1YGP6 A0A089Q204 U7NY76 UPI0003B481E4 W9V341 UPI00067F4562 H2IWS7 UPI000675D9DD A0A098RE99 UPI0004212DE4 UPI0005C15FEC A0A097QPF1 UPI0005D339A3 UPI0004D8E29C E1VCA4 A0A0C4WTU2 E0FET2 A0A0H0Y092 I0QQI6 B7UMN3 UPI00030B6E67 C1DJY5 UPI000248B5E1 UPI0002F588B8 UPI00038060C2 H4I3H7 UPI0006148CBA M9YDT7 J4TTN5 A0A0G9M026 H8NUJ7 H4JUI7 UPI0005B789BB UPI0004E1F9C3 S9YCL8 UPI0002DD07EC A0A0H3FLE0 H4KPD2 S2KK42 W0E158 I2NC81 A0A0B4IM65 UPI000554A929 H4L565 UPI0003674641 A0A0F7K0Q6 UPI00031355EC A0A0A3EMP0 A0A085G3A6 H3KW73 UPI000343B180 UPI00048BF236 E0F2E9 K5V6E0 UPI00041ABFF4 H4LJK6 UPI000376C869 UPI000395D43A E0F8J5 UPI00066B2D3D UPI0003089400 H4IZH1 S5T4K5 UPI00046F29D9 E0EW49 UPI0005F9A9ED UPI0004719479 H4JFJ8 K0C8L7 A0A0F5ARC9 E2P8X6 M7RIV0 UPI00058F6D4E H4K9V6 A0A0F9YVG2 Z5XTJ6 W0Q1J8 K5TSH9 C8NBT1 E3XW38 UPI0004057986 UPI0002AA68F1 UPI0005856421 C9QC04 UPI000660E94E H4IIJ0 D2TWS6 A0A0F4S821 A0A0B5BWF7 A8T649

TABLE 6 List of Bacillus stearothermophilus PcrA homologs that have 50% identity to and 80% overlap. 1747 members of Uniref 50% identity cluster is shown (citable UniProtKB and UniParc accession numbers are shown). P56255 J7M5U5 T0TN09 A0A0I6PI88 R3VBE4 UPI0005CD7F53 S7T032 A0A0H2UUM0 F8LQ03 A5LVX9 E0G4K8 UPI000417C0DE UPI00051815BF Q1JLF2 C2LSM3 UPI0005E41D7E E6GJJ0 UPI0005CD905E A0A098L684 UPI0003C7B0E5 UPI00065FC663 S7YIM5 S4DY07 G7SM20 U2YC97 UPI000254D55F UPI00066E20BD UPI00066CDBC6 C2H162 UPI0004062509 G8N340 Q1J6A6 E3CPD8 E1LG87 R3D1M0 UPI0004051F87 T0Q4M4 A0A0G2V0F7 W3XXV6 A0A0I6BPW7 X6SFW2 UPI000411FB5C A0A063Z1I8 M4YYG1 T0T6T2 A0A0I9JBK7 X6RK63 UPI0004022BA7 L7ZT56 A0A0G4DFH5 UPI0002AEC4C7 UPI0005E02B0B R4CW85 UPI0004188987 V6VMU8 UPI0001E10349 UPI00065FB970 UPI0005DC8263 X6RKD4 UPI0005CE22CD Q5L3C0 UPI00000D9968 UPI0003167399 UPI0005E61B75 X6SVN5 UPI000400A66B UPI0005CD09ED A0A0G3U9S6 A0A0A1DXP2 UPI00066DCA04 R4DBG2 UPI0005CD5B4A A0A0D8BW89 Q48T98 A0A0F3HAQ8 E0Q0Y1 C7WG78 UPI0005BE8F33 A0A087LEV1 UPI00038E29D2 V6Q5R6 UPI00066CD043 C7W6F8 UPI0005CC9805 UPI00066FD17E A0A0H3BYK1 A0A0C2HKT0 UPI00066D16C5 R3EDY6 UPI0005CC91DA UPI000519CC89 UPI00050BF55F UPI0004E153F7 UPI0005E1DA5A E0GYA3 G5L3H4 A0A0G3XVN0 UPI0004BE2C5F UPI000288F7E6 UPI0005E04403 R3L6W4 UPI00040C9E90 A4IJY5 UPI0004F92D8A A0A031IBW4 A0A0I6R2B8 E6FGS3 UPI0005CE3BAA UPI0005CCA9FF UPI00066C9AA9 B1YJ16 UPI0005E64F07 UPI00031E170D UPI0005CCD039 S5YVH0 E7PYJ1 UPI0006AA2516 UPI00066B4226 E2Z449 UPI0005CD5201 UPI0004DF596F UPI0004BE34CB UPI00047948D2 A0A0I7U0N9 UPI0002F39C67 UPI00040A255C A0A0E0T7W1 UPI0004BE2973 UPI000683717C UPI0005DBFF52 E6IN81 UPI000419AA26 UPI0006A962ED C5WGR7 UPI00041CB696 A0A081PQV0 UPI00031A4BB4 UPI00040A186C UPI00017E56F4 F5U8K1 K0A8A5 E8KBG6 S4G3W1 UPI0004221FBB UPI0001D581E8 A0A0E4B7C5 U6BA96 UPI00017C1A3F S4FMR9 UPI0005CDC9EE UPI000424F449 Q5XBW2 UPI00047AE0FA A0A0I8Y7H2 C7VZ55 UPI0004624A9B A0A0J0V9H4 Q1JGI8 UPI000494D4D3 UPI0005E6A918 C7UKX9 F4EF32 A0A093UDD5 UPI00044FEC83 UPI0004792A31 A0A0F2E3V6 A0A0E1C082 UPI0005CF3160 UPI000539F1EA K4Q9V2 UPI000494A958 UPI00066DA31C R1KTX5 UPI000400B1DD N4W917 UPI000617EC21 U7USF2 UPI0002AF45D8 UPI00045B8E48 UPI00040B8112 UPI00055386C0 UPI0003C7BD0F A0A0E0UWM6 UPI00066EE4B6 R2UDI9 B9WTD9 UPI0005590F34 A0A0F5P2U0 A0A0E1R8Q5 A0A0I8ZZI4 R3B3J0 UPI00041EA41C UPI00020D9901 UPI0003C7D5B0 A0A0E1Y218 E0SZL7 R3J9A8 UPI0005CEFECB I8UBH1 UPI0006181569 A0A0B8RF49 UPI00025ABE1B UPI00032DCFB5 UPI00041FE6F0 UPI000555B2C6 I7WIP9 UPI000035D23A A0A0I7UZI3 UPI00032F5E7D UPI0005CE9E1B UPI00059000A4 A0A0G2V4A4 UPI0001B43587 UPI0002313C8F A0A0H1TNE2 UPI0005CED2C1 A0A089XIR5 I1ZL68 UPI0003591B75 UPI00066E2824 R3C367 UPI0004128A2C T0TNV3 E8K3X2 UPI0005128D3B A0A0I8XVX0 R3VC46 UPI0005CF2FFD A0A0H1RMM4 W1Y2G0 UPI0003EC8641 UPI0005E6A956 V7ZS55 UPI0003FA464B D2BQM2 W1VGX2 A0A0H4NBP6 X8KE98 C7YF48 UPI0005CDDD5E A0A0A7T646 UPI0002F353EC A0A097B674 UPI00027EA587 R3W0C9 UPI0005CD2C43 T0W2G0 F8DFS6 A0A0F5Z989 A0A0E7WHF5 C7UTX1 UPI0005CE703E H5SYV7 UPI00031E513E E3ZRG0 UPI0005E9B66A C7UDZ4 UPI0005B9BF22 A0A0B8QL14 E7SC56 UPI0006282029 A0A0I5V7V4 C7WW04 UPI0005CCB173 G6FEQ4 UPI00021BD63E UPI00052F1B16 UPI0005EA0304 R3KHA0 UPI0005CD2248 Q9CGH6 I2NTT3 A0A0H0TBR3 UPI00066ED988 R3KQT9 UPI0003F9DC72 F2HJH7 UPI00065F8C03 UPI0001975CA4 A0A0B7LAG3 U7S5K0 A0A075SIP2 T0V8Q3 UPI00066A6FE6 UPI00003CA336 M5K5E4 R3Y2U6 UPI0005CCB671 U6EMQ9 U5P378 A0A0H3GCW7 M5K8F4 R3ATF7 D5AH63 A0A084A9A3 A0A0F3H3Y1 A0A0H3IUG2 M3IAM8 R1LJ35 UPI0002195DB2 Q02Z69 W1VGW0 UPI000431635A D3H992 R3NBA7 UPI000367B16E U5PKA6 UPI00066BB1D3 UPI000396C49F UPI0005DADCC0 R3PMA1 UPI00040780E6 A2RL58 U5PAT9 UPI00057E5CD1 A0A081QTN0 C7WTA5 UPI00047DE9FC T2F5M7 UPI0004E25F53 UPI000541B044 A0A0F2DL31 C7V916 A0A0D6A4Q8 K7VSC5 UPI0006600922 UPI00065E0E60 E9FH57 C7VLS1 UPI0003079325 T0VAM5 UPI00066E3612 UPI00059B4903 K0ZJQ5 R2VJF4 W1SU53 UPI0006172C2A V8BFU1 UPI00064CDA40 UPI0005EA108D C7VRD8 K6DTK1 G8P2F6 UPI0006605290 A0AJL2 A0A0I8KXT3 R3I2U9 UPI00058D7294 S6FGR2 UPI00066EBF4B UPI0004F3EE62 G6R9C9 R3I1C1 UPI0005F025AA UPI0005838B59 F9M2F0 UPI000570B4C0 A0A0I8MDZ1 U7SAR7 UPI00055EBF2F T0UFT5 UPI00066C8E1F H1G7W7 X8HMR1 R3DJQ9 A0A0F3RNZ7 Y1QH73 UPI00066A3914 UPI0004D59A43 J4Q880 R1JZF5 UPI0005534922 UPI000629DD6D UPI00066CB902 G2ZBE3 W3XTS5 R4ALC3 E7FSU1 Y1Q968 E3CEE7 A0A0G2WHH3 UPI00066C1FD6 R4B5K9 G2SLV9 UPI000376C4D7 UPI00066DB632 A0A0G2W8X5 UPI00066BE89C Q837V7 A0A0G8G5N5 A0A0D6DXQ0 UPI00066D30C4 Q92AP9 UPI0005E8CD89 A0A0E1RBS1 R6S5W5 R2SEZ2 K8MRD3 Q8Y6C9 K1ABC3 A0A0F5AWZ1 A0A0G8G9A4 UPI0003F7ADC6 UPI00066D94ED A0A0G2VQM3 K1AGK6 A0A059N1T2 A0A0B4B1V1 F9DT42 UPI00066CDADB A0A0G2VT50 K0ZZ87 S7U796 F7R373 UPI00054EA513 A0A0F3HXT7 UPI0001EBB745 A0A0I6M9Y8 D4EPU4 UPT00062CB1C4 UPI00030811AC I7IYF1 UPI00066D78F0 UPI0005E36939 U6RZD7 UPI000409F2F6 UPI00036C3403 UPI000345D943 E6J2G2 UPI0005E225FD E6ISD3 C0WZU5 S0KSL1 Q8ES83 UPI0002FD7F0C A0A0I7LKZ4 J6NDX6 A0A0G9GFB1 A0A0A5GAU1 R2R7H3 W1TUK9 A0A0F2DPD4 S4GCT9 V4X193 UPI000414CEF0 R2VFE1 I0S6A8 UPI0005E11B6C C0X2D5 A0A0F4HGR5 A0A078MEE0 R2RQ91 UPI000660E090 A0A081QG94 J6PVS6 U2HDV7 UPI0004103FCF S0RUY8 F5U2X9 I0SZR9 J6ESY5 D8IIP0 M1Z7Z9 UPI00051A74A9 U2XKT4 UPI0005E14969 S4C6D0 T0SLF0 UPI0001850A35 W9EJ23 K8YYY0 UPI00066A9D62 J6GZ70 D0DT13 J1HTP8 UPI00028D8F83 T1ZV68 A0A0E8P896 E6H2K6 R4RJL7 F0ELG6 K0IW42 A0A0E2IST8 UPI0005E5529F E0GC91 UPI000582B547 UPI0002E3189C UPI00059D08F4 I0SM41 A0A0B7M600 E2YA71 UPI0006657CCS T2NS83 E5WTD2 UPI00066B626D A0A0I8X1A0 S4DTD6 B2GDT0 UPI00042432D9 UPI00047A5B10 UPI0003906A09 UPI00066A9CD2 E0HHS0 A0A0G9GHB4 S4E2N9 A0A0J5YRT7 J5H823 I0STF6 J6CZA7 A6CTQ5 UPI0004065D0D UPI0006A9BD02 F9P5H7 UPI00066EB944 E2YXC8 UPI000468231D S4B0D0 UPI00031F0DBB UPI0006612AD9 A0A0H2UPX1 J6F8R9 A0A0A2TG25 UPI0004080059 W7KMF0 UPI0003908243 V8IKB2 E6HPG5 K9ECC9 C8ZW14 G5K2S6 UPI00066D6CAA B2IQ19 J6LKR6 UPI00048127F0 G5ISN9 I4X463 G6A4N0 A0A0E7X876 J5ZVI4 G2KVV1 R2PGJ0 UPI00052FEEE4 UPI00023296C1 UPI0005DD5C54 J6QD02 G5KC28 C9CH47 A0A098EQM7 UPI00066A46EE A0A0I9AQP0 J5DL74 F3L6C7 C9A7W5 W3AD61 UPI00066A7CBF S9RFX3 J6EQP8 UPI0003106D0B T0UB13 A0A0B4RAA8 T1ZDX9 UPI0005E228BC J6NTK1 UPI000419C333 A0A0C2Y4G1 UPI00069FBE5D X8HC25 UPI0005E426DE J6PAC4 UPI00047C345A A0A0D0Z5K2 F6CR29 A0A0C1HWD2 UPI0005E7453E D4EZ78 UPI00069F1374 B4U2N2 G8PE15 UPI0001F606A4 S7XP08 E6IB45 A0A0J6BIJ7 UPI0005BB133A V7HVC5 U2YEQ3 UPI0005E54BE5 E6EW25 UPI000345FA17 C0MG96 A0A0F4LU79 UPI00066AA721 UPI0005E4BCD8 J6LYS0 C0Z4D5 A0A0D0ZF61 C7XVU9 A0A0E9EVC1 A0A0B7L333 E0GI65 L5MLM9 A0A0E1DXS1 F6B7U9 UPI00066A249D R0N6B7 C2DEW5 J2ICT3 A0A0D1AJJ8 UPI0001FAE64A A0A0H4QJH6 A5MXJ1 E6HGC1 J2GML2 UPI0005BB6148 UPI0004119D25 A0A075QWT2 UPI00066CEE14 E6GUV7 UPI00036424CF A0A0G7A7Q1 E1UN03 UPI0003821CF7 UPI000352C53C E2YIL6 A0A0H0SII0 UPI0002175097 A0A0A0TU62 H0UC09 A0A0I6M0R5 T2P3G0 UPI000673AC8A C0M8W6 UPI0006480DA4 UPI0003B19331 J0UC50 J6R7F3 UPI00037AD71B A0A0G6YRU4 I2C272 A0A0F7BZE F9PC08 J6QBF0 D6XYW2 A0A072ELK0 A0A0E1LP36 UPI0005556E3B UPI0005E65946 T2P2M8 UPI0005A0C938 UPI0005B9A818 UPI000458757A UPI00029B0B59 UPI0005DEEC80 C2JPD9 K0NSF0 N1ZT17 UPI0002458DDC UPI0005A8DE74 UPI0005DB5FE3 F3R2R2 K0NWD9 A0A062X6V6 UPI000471D8DA UPI0004936DE9 UPI0005DB0E2B C2JPD8 I7JV33 UPI00046A8E16 A0A0G3V8X1 UPI0003104D4F A0A0E9A2L0 J6F4X7 UPI00039E5A1A UPI000219434D A0A0D1IMY3 A0A0G2Z1V7 G6PSM2 UPI000665D580 UPI00066064E2 UPI00054D6FB4 U1YND2 A0A0H1T5V3 A5MAN0 UPI00053BEB7B UPI0004959EB5 Q5FLL5 UPI00069E39D3 UPI0002BAE34A H7JF57 W1VWC1 UPI00020DA0D4 A0A094K9S3 UPI0002F6BFBB K0JN87 D6ZSN4 UPI0002FDF1DC M7NB45 UPI0006735FF0 UPI00028966FC UPI0002BAFF71 UPI00067E3BD1 UPI00032EF110 I4D367 UPI0002F19FB8 UPI0005F96F19 UPI0002E84A37 I0Q912 UPI00032ED949 A0A0J1FQT F8DMZ0 A0A0G2YPJ5 A0A0H1VSP1 J0VYU9 UPI00032DF268 UPI0005A135DF A0A073K223 M4KP62 A0A0H1VST5 A0A0I7N7X2 UPI0006611725 UPI000557D9AE A0A081NN78 UPI000467CBC2 A0A0H1XJQ7 B2E1F7 R3KQ79 UPI00040DC1E5 UPI0002DA8E1F UPI0004E7B7B4 S8PCV2 G6S5S7 UPI00032D8816 G5JVH9 S5N6B2 UPI0002A11EAA S9B2B8 G6LMK2 UPI00032F27BE A0A061N7C8 C0YY61 A0A0G2LYN9 F8Y0Z9 C1CE74 UPI0004244B9B UPI0005525FE7 A0A0H4L928 UPI00046ED5B5 S9MTU2 A0A0I9HKT6 R3JUC0 UPI00034C8C5C A5VLG7 UPI0006529B7C S9CV37 A0A0I9C672 UPI00045A0DF5 D5D9D8 R9WGA8 L8PVW0 S8FGZ3 UPI0005E5C16F C7CXQ3 G2RXJ7 F8KEB4 E0TU76 S9BAQ5 UPI0005DF7A6C I7BW34 A0A0H4R638 B3XPD6 A0A0D5D2X4 E7S415 UPI00066DA9B2 A1HME8 D5DWY1 A0A0F4LTY8 UPI0002B6EC1D UPI00046CB8EA A0A0I7G3B6 Q5WJ31 A0A0F0J8P4 UPI000487320D UPI0002891107 UPI0005E2BE93 A0A0F7Y925 UPI0002FD32D5 A0A0B6AB4 D5WUX8 D4G620 S8FZ09 UPI00039C59B5 UPI00067A2901 UPI0005B4512E UPI0002F118C8 UPI0005AD3458 A0A0H1GG85 A0A0I5N723 S2XF35 UPI000682E43B UPI0001E89712 UPI0002865556 Q8DZF9 A0A0E7ZAF0 UPI000679FA7D A0A0J5USU5 A0A0J5GSJ3 O34580 A0A0H1WCN1 UPI0005E318B4 A0A0A8HNC8 A0A0J1IVB0 UPI00068A6D6D G4EZ92 A0A0H1YVD6 A0A081PXD6 A0A069F1G6 UPI0004733E51 A0A062XR18 A0A0A1LU02 UPI0002BE4DD4 UPI0005E614FB A0A085UDQ4 UPI00047C38B4 UPI00021964BD L8ADL0 UPI0005DE829A UPI0002735B06 UPI00036BDB49 A0A0J6WUD7 UPI000415247A UPI00046730CE A0A0H1IDA8 A0A0I5WGP4 A0A094WEU8 A0A098ESM7 V6Z3H5 I0F1C0 A0A076YVH2 A5LMH1 A4J705 UPI000423B330 UPI0004205ED9 UPI0003A70724 UPI000332E0DF UPI0005E59371 UPI0006527050 UPI0003882373 UPI0003817B64 UPI000416C8D9 A0A0H1LVT6 UPI0005E5CDC9 UPI00053C5AB9 A0A024QGT6 A0A0J8G63 A0A0F5MHF6 UPI0002B9E962 UPI00066E2A51 A0A099YFE2 UPI00036A8D74 M3GSH8 UPI0002A13E19 UPI0002B9AAC1 UPI00066DCBF7 A0A0D4CNA9 H1LDA8 H7F3P6 UPI0002416823 S9MD09 UPI0005DAF614 UPI0003FBD3F6 UPI0002E51DEA W7DTL5 A0A0F0AC16 S8NNN7 UPI00066C80E0 UPI000378183D H3NEA0 G0UES1 I2HNA7 S8P6Y4 K8MSF0 UPI0002E4B790 UPI00058BA923 C5RBQ7 UPI0002E6650E S8QFS6 C1C784 G5KGH3 A0A0A3TV65 D7UU99 UPI0002AA99BF A0A0H1NAH8 UPI0005E0C55C A0A0F3HRU1 K8E0T1 W7BIE4 UPI00057C2EC9 UPI0002F54753 A0A081QQF8 J7TJ76 UPI0003F5E34D W7BQB1 UPI0003872E1B UPI00028CE135 A0A0I6RW2 F0I8F9 A0A074LMV6 A0A091BQ28 A7Z260 UPI0002BE2494 UPI0005E787E3 F2CE01 W7CP40 B1SEW1 S6G180 A0A0H1HP61 A0A081Q205 E8KQC7 N0AL42 UPI0005C4C4A5 UPI000398030A S8P0E2 UPI0005E3629B A0A0F2CZP2 J0L6J5 S5RL61 UPI0003A660B0 Q8E525 UPI00023100D2 F2C6S2 UPI0004915921 UPI0005C46508 UPI000345B919 A0A0H1DLQ4 UPI0005E1CE26 N0CB15 UPI00035FC8AB H6PBJ7 UPI000665EEF6 UPI0002E871D3 A0A0I7RCB5 A8AXV7 UPI000525E54F UPI0005C676D5 A0A0D7XRK8 S8VI39 UPI0005DADB38 UPI00066BA6D4 R4KJT8 T0UQQ7 UPI00038048E7 UPI00030AEE41 UPI0005E20280 D0RVG1 UPI00024669AD A0A060RKS5 UPI0005A43216 K0JMH9 A0A0I6RJI8 F0I0L3 A0A0A1IK06 F5X182 UPI00057BD0DF UPI0002B8C27C B1IBM7 F0FSG1 Q9S3Q0 UPI00066000C4 UPI0005EBB97E UPI0002F07C72 UPI0002ADCFE5 UPI0006B25214 B1MXQ7 F5X6S0 UPI0006AE6F38 A0A0H1Z5L A0A081QYR3 UPI00066A525C A0A098BA44 E0PDX9 A0A0H3DY38 I7ISP9 UPI0005DF6E72 A0A0F2CRI4 H1WSK1 A0A0E1XF14 A0A080UNJ0 A0A0H1T3F9 UPI0005E41938 A0A0B7GK35 A0A069CV32 H2A6M7 UPI00022BA657 A0A0H1MS35 V8IC09 F2BSX1 UPI0005F00C2A A0A081JHH2 A0A0C2PX57 A0A0H1V4C2 A0A0B7L5V0 UPI000660334B A0A081BK49 UPI00051C02C3 UPI0002DC92B0 S9APZ9 I2J4H2 UPI0006616F5F A0A0A3HZB UPI000410E728 UPI00066FBF08 S8FYF6 A0A0I7VHR8 F0IU30 F4BLW5 UPI0004D4E3B7 G4NUA0 S8HPX5 UPI0005E06D6A A0A0F5MJY5 U5SAC6 A0A091BNX2 A0A068LM65 UPI00046C8FD9 UPI000318BEDD F3UWM7 A8U4Y8 E8JNI7 UPI00025F1BD8 K9AHW3 A0A064BXT9 UPI0006603308 UPI0005590D06 UPI00040D1DBE I3DZY5 UPI000554B661 A0A098ZS00 F0INA4 UPI000550B1B9 UPI00041D9E3E A0A0F4M491 C2E4W5 S2UT89 UPI0006605CF8 UPI00054D85C0 W7ZBP8 UPI0005145490 UPI0002FE2AC2 UPI0005DBEB5A UPI0001FBB9D8 UPI000553C539 A0A060M0P3 A0A0F4LJ28 A0A087QEX7 UPI0005E44ED7 A0A0F2CJE0 UPI0005C4A619 V6M8R1 A0A0F4LIP7 UPI0000459CDE UPI00067A8E24 UPI0003D2BF73 UPI0003B36792 A0A075LMV5 A0A089Y3J1 A0A0A8B472 UPI0005EA38C1 A3CM74 UPI0005CE4434 U5L6Q7 A0A0F4LHL3 UPI00057E310A UPI0005DFA93B UPI00066124BC I7LCW7 Q2BBV3 UPI00066D84FA F7SF58 UPI0005E43F38 UPI000204C9B3 UPI0004942515 J9YKR2 UPI00047168BE UPI00069ED3EB UPI0005EA228D E8JTX8 U1N3Z8 B9E831 UPI00031D0F4F D0R5K3 UPI0002D988F7 F3SII1 UPI0004DFB32B A0A0A5GC60 A0A033UAP9 Q74I48 UPI00016C30E9 F3UBI9 UPI000495DE5F UPI00068F9B5A UPI00065B9E05 V5P429 A0A0E9GYK1 F0FE53 C4L2B1 D5XD67 UPI00030B5B12 F4AC33 UPI0005DF5506 F9E172 A0A099DEL3 S4D0X9 R2S3I5 UPI000660D235 UPI00066C8BEE F9HGV1 UPI000554029F UPI00040BD27E UPI0005BB07E7 UPI00066974FD D2ER82 F3URE9 S3FYE4 W4QBS4 A0A0D1LT71 UPI000665A968 G6JAI2 A0A0A0DGR7 UPI0004787EFF UPI0001E98CB8 A0A0D1LUL0 UPI0003421E15 A0A0I8YKR7 UPI00066B8271 A0A069EVD4 C8PBZ7 UPI0002191623 UPI0001A57BCC A0A081R5K4 W4F985 UPI000426A771 UPI0001E2B095 D8MGK9 D1YI75 A0A0B7MAG0 UPI0006A9F3B8 S2KHH9 UPI0001FDB18A D5T1C2 UPI00050F2122 A0A0I9KK18 UPI00052475A3 Q1GBF8 UPI0002072E3B F6DPJ5 UPI00050F4C6F J1DH18 A0A087N1S5 UPI0006800765 A0A0E1XZC6 UPI0002E9BB91 A0A0J7K5W0 V8I629 A0A078MFK8 A0A061C4M6 E3BTD7 L0E9U7 A0A0J7HHM3 UPI0005E3DD18 UPI00037E072F E4SWU2 G3YXZ2 UPI000401406C D3L7Q3 UPI00066D82D8 UPI0002F64287 D8FRH5 E1NV43 A0A0A6NWI1 Q04GN7 E1LLK4 UPI00040FF648 F0HX91 UPI0001FD875B UPI00047166B2 UPI00050F2BFE A0A0I6S0N2 UPI0002FBC45B Q71HW2 E1NLU9 A0A0A1MVS7 UPI00050DE508 I0QC96 S2XYX9 UPI0000510635 UPI0001E5D8E8 UPI000595B757 UPI00050F1464 UPI0005DCF59C A0A0E4H7N7 A0A061CFU6 A0A061P6S2 J3F9X4 A0A0E2VR29 A0A0D6J8D1 UPI0004758748 UPI0006829E76 A0A061NMB4 UPI000410FF91 A0NL49 UPI00066CDBD2 T0BRJ1 F0K0A9 UPI000364DDD7 UPI0002F4C1DE UPI000277B8D1 UPI0005E4C005 UPI0004CECB95 A0A061BVE6 A0A0A3I330 UPI00046A2EE8 UPI00050FB3CA J5GJF0 UPI0002DA0189 G6F4X4 A0A0A3J757 F0NVP6 UPI000277B786 UPI0005E2E74F UPI0002E0B711 UPI00032F7094 UPI0003107DC7 UPI0002DD6873 UPI00050DA477 A0A0E8AXP8 UPI00037D657F A0A061CH35 UPI000465B065 W5XDQ0 UPI000277B3EF F3VID1 UPI0005B651F3 A0A061BSV4 UPI0002B59EE5 UPI00046C96B8 UPI00050EBFE8 E0TNB3 UPI00031B78EB A0A061C0Y8 UPI00031ED71B C2KB80 C2EVD7 UPI00068136F1 UPI0002DA8172 G6EVU1 UPI0004225966 UPI0002E9360C UPI0004953241 UPI0005DD3E82 UPI0002FE6543 UPI00069A32A5 UPI0002B55DD2 UPI0001B2ADB8 UPI0004256505 A0A0I6F783 UPI0002EB9E28 UPI00034B89F3 UPI000268A7D1 D5H1T9 R9TUH2 UPI000680414A UPI000362867B V4QC95 UPI0002DD3475 V5E0D8 A0A0J6H039 I0Q427 E3CGX4 UPI000287FE90 UPI0003140F32 U6FUY0 A0A0D7XCT2 UPI0005E1E1D9 UPI0003675904 UPI000493F21F UPI0002DF577A U6FDW7 A0A0D7XAM9 F9HM88 UPI0002BE1554 UPI0002F64068 UPI0002B52183 U4QEF3 UPI000470EB15 R0NC47 UPI00036D9746 G9ZMU8 UPI00030AAF28 C2ELL7 A0A0H4X8R6 UPI0005E3138C UPI00031BD2D4 I7JG80 UPI0002B54D20 F6CEU6 T5HV53 F9HDZ5 U2KML1 UPI0002490095 UPI00030ED4DD A0A0D5MK99 Q65MR3 UPI0005DF6040 UPI0002F0FB31 A0A069DTV4 UPI000317D564 U6F2P2 UPI0003A92B3A UPI00066DF5D2 UPI0002DF3A61 UPI0003718539 UPI0002B5AC18 A8YTZ2 M5P7T2 UPI0005E78EF0 UPI000309D239 A0A0A5GG76 UPI0002B53160 UPI000468D9FF A0A0F5KXW3 A0A0I7SUM9 UPI0002E4AADA UPI00040E6B55 UPI0002B5578F UPI000698FD57 A0A0J6ENX9 UPI000399E547 UPI0002EFE60F UPI0005CD2072 UPI0002D5DE55 F0TDD1 A0A0J6ES4 UPI0005E5D243 UPI0003714975 UPI000487D20C UPI0002FFCB1B F2M355 UPI0005A16A5C UPI0005DBEC58 UPI0003773AA6 UPI0003720301 UPI0002B50165 R5ZGC9 H6NTK0 UPI0005E3FBD1 UPI000375ABD2 M3HNA0 UPI0002B5E93C E4SM94 F8FKW5 D4FT83 UPI0002DE0320 UPI00020CBEE6 UPI0002B560C6 C7XLR3 UPI0003F50A86 G6CA70 UPI00035FC09C A0A0A6UX77 UPI0002B58B2 J4BVM3 G9WIE0 A0A0B7LL60 G5JQP7 A0A0E2USS7 A0A0E2ES03 D0DI93 UPI00054F4F52 R0MM09 UPI0002E29189 F1Z0K5 UPI00030C4A19 K1MXE4 A0A090IVN1 Q8DPU8 UPI00037CA55E UPI00041B294C UPI0004648E2C U6FHU9 A0A0D0FW99 A0A0H2ZN88 UPI000379270F UPI000513CEDF UPI0002B588C8 F3MR07 A0A0D0FHN9 A0A0I5SPG K8Z8B4 G0VS69 UPI0003111877 W7YRH9 A0A0D0F833 E0PRK3 A0A087EPN1 UPI0004221C8D UPI0002DA340C W7Z816 UPI0002195446 UPI00066AC65D A0A063B7B8 UPI0004858886 UPI0002EE7FD7 V6J250 J2ZKY4 I2J7J6 UPI00054DF8E8 UPI000356EF52 UPI0002D4555C C0WSC1 UPI0002192DE8 F5W0L7 UPI0006B251F7 R7MZQ6 UPI0002B513D0 C2D3L0 A0A0J1HWV3 S3BFG0 UPI0006B25740 UPI000550AF6D UPI0002B5E0D9 C0XHM8 R9BWL2 E8K074 UPI0006B25166 A0A0B0IKT7 UPI0002D9F995 F5L444 A0A073JY52 UPI00066B28A6 A0A0C2WKV7 W4QVT6 UPI0002E8D24F A0A0G9MI18 UPI000482AB97 J1S8C6 K0DDF5 UPI00054D3339 UPI0002B58C84 Q03Q12 A0A0E9GQ05 UPI00066E5C86 S0NID8 F9VEL8 UPI0002B53813 U2PJH7 UPI0002D32EA8 F9PWL8 UPI0003F6F988 V8ANU3 UPI0002B5E4B1 M5AEL4 G6LUK0 UPI00066E06C6 UPI00047D412F UPI0006226EE6 UPI0002EFF9CD A0A0H4QAV4 UPI00067AB63B UPI00066CF5FD UPI0004016A4F UPI0002D66EB7 UPI0002B58C9A UPI0005B64156 A0A0I8X5X0 A0A0F3H5E8 UPI00040AD781 K2PIY1 UPI0002B58360 UPI0004882072 UPI00066B62FD UPI00066B0571 G7SH52 A0A098CN34 A0A0E2EHS3 A0A0C1PU47 UPI0001DDD26E A0A0F2DYG6 UPI0003FB4C2E UPI000266D5EF UPI00030C12E8 UPI00041E7D06 UPI0005DB8FEA A0A095ZC63 UPI0005CE1998 UPI0002FD1A27 UPI0002B55BAC J7LCK3 UPI00066EE137 A0A081SAK2 UPI0005CD605D UPI00031F2D35 UPI0002B4E249 A0A0A7U0Q8 I0SAQ0 UPI0005342342 UPI0005CDF885 UPI00031F5691 UPI0002EC6AC7 UPI00068191BE F9MKL8 A0A081Q0K0 UPI000424E658 UPI0005AA507F UPI000466520E Q03YP2 F2QD74 UPI0005358034 UPI0005CCF850 UPI00040E6C2A UPI0002B5A6DE UPI0005A1E84F A0A0F2DG50 UPI00067A78F3 UPI0003F512C0 UPI000472C523 UPI00046753B6 C2KL56 UPI00069CF310 UPI00067BFD62 UPI0005CCF8E5 UPI000373D4CE UPI00031B4772 T0VWY9 A0A0F2D2Q6 H7QP48 UPI0003FB972A UPI000672387D UPI00046312FB UPI0002341443 UPI0005EA00F8 J1V408 UPI0004079443 E7RJR7 UPI0002D7CB42 UPI0005A6A464 A0A0I8VWS0 E1MA03 UPI0004030821 A0A0A2UWH4 UPI000466DCC8 UPI00046CA6CE A0A0I6VM15 UPI000411F965 UPI00040E7BC1 UPI0002880C4F UPI0002B550C2 UPI0006814228 A0A0I8S594 UPI0003751A95 UPI0005CD07D9 A0A075TVF2 UPI0002B59082 A0A0H4N937 A0A081QA48 C8P6C2 UPI0005CDB669 UPI00054E004D UPI0002B5EB7C A0A095AHY9 A0A0I9J5Y7 UPI000312AA9F UPI0005CEAA59 UPI0005A65B8B UPI0004633B74 UPI00037F25A9 M5N794 E3CAD3 UPI0005CE6576 W9ANP4 UPI000264EF92 B9DUG5 UPI0005E67219 UPI00021A3A73 UPI000409F746 M9LGB1 UPI0002E7D773 UPI00062028C1 G0I9B0 K0U2M0 UPI0005D16915 H3S9K5 UPI0002D3B5B3 UPI0006203F88 A0A0E2P693 U1ES89 UPI0005CEBA00 UPI000378CC96 UPI0002B577F4 A0A0F5I4J0 F9NZH5 UPI000660DA3F UPI00040F513E K2FNB3 UPI0004662568 A0A0F5HRS0 UPI0005E921D9 C8WTI7 UPI0005CD9361 UPI00067F085F UPI0002FBB47E Q5M4H1 A0A0H5LNP6 B7DQB4 UPI0002322E59 UPI0001E2EBD3 UPI0002B5379E UPI0002DE5460 A0A0E0X8J2 F8IDV6 UPI0005CF78F9 UPI0001FDB5AD UPI0002B51579 V8LWU0 UPI0005E64E83 UPI0005599D20 R4NWS6 D4W731 UPI0002B4E9DA E9DMH4 F9LXX0 UPI000509968E UPI0005CEAB2F UPI000490D40D UPI000464DB1E UPI0003121E5D X8K6Y7 UPI0005CABE96 UPI00040E0B64 UPI000255C522 UPI0002B529E6 J7T7E4 I0T8M4 A0A0J5S290 UPI0005CD124C A0A0F7D4N3 UPI0004671B60 UPI00031AFBDD UPI0005E22D3B A0A0J5WFP0 U5UIJ5 UPI00058E169D UPI000319EA31 E8KV65 E1LS08 A0A0J5YA29 UPI00042996E4 UPI000624F4AA UPI0002FFFF03 UPI0002E8600B A0A024DEK7 UPI0006A9C586 UPI00042A2929 A0A0A8JEM1 Q8DTY6 F8HD36 UPI0005E2SBC5 C8NHG1 A4VUA8 UPI00047BFF10 UPI0002B59757 A0A0E2QHQ8 UPI0005E30B11 UPI0005874702 A0A0H3MVK6 I9B3V6 UPI00035CDC0C F8LX97 E1M4S7 UPI00066C1DCE UPI0005CD2519 A0A075K9S A0A084GLL3 A0A0F6BVJ6 UPI0005E14CCC D4YVQ1 UPI000409C6E9 I9NQ12 A0A084H1D9 A0A0E2RHF6 F5VXC9 E6FS51 UPI0003FE3351 UPI0004883363 E6TWN0 UPI000264F340 A0A0E8T7V0 UPI0002EA5AD2 UPI00041E695A S4NRZ4 K1LG40 UPI0000E563DC E6KMR2 S4CP69 UPI0005CDA05A J9W320 F2F7J1 UPI000660EC4F UPI0005E76F14 UPI0003FECF16 UPI0004038E95 F4FSH6 J1GP52 UPI00066C13CA UPI0005E0C70E F2MQT5 UPI0005CE89C0 UPI000403AE07 F8HYK0 F8LIZ1 UPI0005E6F0D4 UPI0002A3D37C UPI0004018E0C A0A084HBI0 UPI00044D3C3A G2GTJ2 S7YYN6 E0H8L5 UPI000404D8AB D3FTF3 A0A0C6G2S0 X8J9A0 A0A0I8TLZ0 R1W0H5 UPI00041CFDD8 U6SL82 U2W3N6 UPI00066AA528 E9FJW6 S4FW64 UPI0005CDED2A UPI00036426F2 A0A0E1ENC5 A0A074IU47 UPI0005E93C3F R3UP49 UPI0005D236D1 UPI00047A28D8 Q99ZE1

TABLE 7 List of E. coli PcrA homologs that have 50% identity to and 80% overlap. 1029 members of Uniref 50% identity cluster is shown (citable UniProtKB and UniParc accession numbers are shown). P03018 K8BG21 UPI0002C8F355 UPI0005A9630D UPI0003EF5338 A0A0J0DJ77 A0A0G3HMD3 A0A060VDV3 V1HN20 A0A0A3YR40 UPI0001F6648A A0A0J0SUX3 U9ZBE3 A0A0E1CLV1 UPI0002C9B17D UPI0005EB7A8B UPI000678B341 A0A0J0M6S9 A0A071CB77 W8V249 UPI0006811593 UPI00058E54A4 A0A0K0IDG2 A0A0F0XZS7 S1J559 A0A0J2G3Q6 A0A0J4VXC9 H1C573 UPI0002CC80BD UPI0005D0A9E9 V2S4E7 A0A0H4Z3E1 UPI00025C7C5C UPI0005CD86D3 A0A0J5K2Q0 A0A0C8UHF8 A0A073G662 V0AU35 UPI0005304A96 UPI00044E7286 A0A0H3MJV2 A0A0C9HTD3 I2SQY0 A0A0H4YPU3 UPI0002CAB12A UPI00037EE7F7 A0A0E0VDJ7 Q8Z3B0 B3X3W4 A0A0H5AHT5 UPI0005CCA08F A0A0J0GVC5 A0A0G2SID2 A0A0E7LC59 N2GY76 W1HG62 UPI000330B244 A0A0H0CXK2 UPI000542989F W6J799 W1F3C2 A0A0H4ZLF1 UPI0002CCBAB8 V3PV69 A0A070RYI3 UPI0004DA823D E1HNQ6 W9BQA0 F3WPX7 A0A0D1KFS4 H3MUW4 V8MJC9 A0A070SNS2 A0A0H3GGJ9 A0A0F6YD20 H5V6H2 A0A070H7E9 UPI00049F5927 H4URJ5 A0A0K0GRR7 K8DQF9 D7YBR7 UPI0003BC8E89 N3EUQ7 M9G7C2 A6TGJ6 K8C9V5 UPI0002C8B609 UPI0003910486 UPI0004693D87 N2IIQ0 A0A0G8G1B7 F5VR52 UPI00063CD924 A0A090UJD6 UPI0002CCC2BC S1HRC3 UPI00058FD925 A0A0D1QDQ1 A0A0C2AR33 UPI0004977D3D F5N8N5 D8E9M5 UPI00058F49FC A7MQJ8 M9I6S8 UPI0003EF3FD7 UPI0006A5855E A0A074HPP5 W1HTQ0 V5U5I0 N3K330 UPI0002CC54C7 Q83IW7 L2VEY2 W0ZY91 UPI0005187950 I6CD07 A0A0G3S4T9 A0A0C7MG10 K5CJK9 F4T661 K8D2A7 E7SHD7 A0A0H3HA95 A0A0G3KPN2 D7YG58 F4V8D3 UPI0002CA0405 B2TUW9 A0A0E0WSN3 Q0SZ04 W1BJG9 F4TMH3 A0A0J0I5H8 UPI000390185B A0A068H452 D2ABY6 N2QEY3 A0A029LAE5 UPI000579149A UPI0005EEDAF9 UPI0004A0FDEC A0A0F6MJ85 A0A069YVJ3 U9YHH0 UPI0006650689 B5RFP5 A0A0H0GX62 F5P1J3 A0A070Y0G0 A0A080IB93 N2J8A4 UPI0004733206 UPI0002CB804F A0A0F6EK00 A0A073GWJ7 A0A083YZ93 A0A063XKV2 UPI00026721AF UPI0002CB6B71 I6BAB7 V0RR87 UPI0005C48DC6 UPI0005C63608 UPI0003A80309 UPI0004D7856B UPI00050B7641 V0ACC7 UPI0005A8BF01 E7T4T6 A8ACW1 A0A084ZTZ9 UPI00050B2FF7 N2RS67 UPI0003710649 A0A0G2XIC2 A0A0A5IRH8 A0A062Y212 I6FW66 A0A069XHA8 A4WG04 I6DJM1 A0A0F1WNC5 A0A064DKM1 UPI00067F497D A0A079H1K8 A0A0J8F6L5 K0WUD4 V3DAP7 A0A080EWZ9 UPI000530716C A0A074IWT6 UPI000666003A E7TCS8 A0A0E2K1D2 UPI000668F9A7 A0A0B1RCP6 F8XAY4 A0A0I1EMQ9 Q31UH5 UPI0004D8D514 V5AU63 B6I4F4 A0A074HJR7 A0A0J5U9E7 A0A085HAH3 UPI0003EF42B5 UPI0002CC06C9 E9TMV0 V1BCC5 A0A0J6MG09 A0A0J5L085 UPI0005A87CA8 A0A0B1FRQ9 UPI0004D72F99 A0A080HWB3 UPI0006684F9F I2BE57 UPI00016A0FB4 S1FP27 UPI00025ABCDF I2X3X5 UPI00058D9C39 A0A0F1BI78 UPI000496CFDD S1L396 UPI000627F480 A0A070FA84 A0A0A5RML6 A0A0J0RXX3 UPI0006ABEED8 S1CI55 UPI000326F8B9 L3K8J5 A0A085ITJ0 Y1GM95 UPI0004646130 L2VN93 A0A0F4HLT5 A0A080GHX3 A0A038CQJ1 E8C7D9 B7MR33 A0A089U9W2 T9FRL3 A0A073FPS6 A7ZU18 V2JXK2 A0A029IIQ6 D2TV17 H5E8S0 S1GRU8 UPI0002CA1DFD V1LV18 A0A029HFI5 UPI000667BF5F I4S2D3 H5J8D4 A0A0F3LUY4 E8D343 A0A0J9KSZ0 UPI0006207A91 V8FG33 D8ERJ1 UPI0002481DE4 S4INC0 A0A0H0KN67 I2X271 I2RVR0 D6I369 A0A0J8LYC2 E7ZSL5 UPI0002CB91AD A0A0D6IZH2 A0A070D8G2 A0A071CFC4 A0A0J8MSQ3 A0A038D0Z4 C3SKC2 B7MH77 A0A026UZE9 M8SKZ8 A0A0J8HX73 E7YUD9 A0A0H8C28 A0A0E2KYP4 A0A028CBA2 S1EV38 A0A0J8QFU8 S4J0L5 V0VC55 UPI000512AED4 V0U5F8 S1CHB8 A0A0J8KFX8 E7YT71 V0SS57 UPI0002C8F6BF H5A4E5 I6CYG1 A0A0J8IWX2 E8F002 T8ZCA9 G9YXY2 G2AN47 H5IRF1 A0A0J8NMQ9 E8EDF3 N4MZW5 UPI0002CC829C K3QIJ3 I2WCK0 A0A0J8JFF5 G5LW37 A0A070K8G3 UPI0004B001CB A0A070SY69 A0A071DAV1 A0A0J8M4K3 E8FVB6 W1BBJ0 UPI0004E37056 I2UC63 A0A070DJ71 A0A0J8HJM3 E7VG83 V0XWV9 UPI0002C98364 M9EF05 A0A079D807 A0A0J8M7Y3 E8AMN0 L2X7H9 UPI000267F8CE A0A027TGT7 V0YB46 A0A0J8LY21 V1PEK5 T8JFJ4 UPI0002673104 A0A0E1SZY6 D8AZQ6 A0A0J8K6V3 E7ZUE1 T5TRC8 UPI00066D844D A0A0E2U398 L3IME4 A0A0J8JD30 E8B3Y5 H4I3L1 UPI0005083EE7 A0A027ZJG3 I4J587 A0A0J8KYA0 G5PV87 A0A0J3V9C5 UPI0002CABCFF C8TL04 T9CEL0 A0A0J8M2A9 T2Q2W7 N4NRN6 UPI0002C94803 A0A028E3K3 A0A070ULP7 A0A0J8KZU2 E7VUY1 U9Z163 UPI0005309A93 A0A026HN93 H4V876 J1GHE8 E8BI66 X7NZ16 A0A0J5MIB1 A0A025G7T3 F3VD13 UPI000472C058 S4IB83 S0YT63 UPI0002CC9136 K4VZX0 K3KG98 UPI0005F8A7CD E7XYR0 H4JUM1 UPI000269547E K4XMA4 G0F7H1 UPI0005ED3E27 V7WD74 A0A073H2N3 UPI00034730CE A0A0H3XBG3 E6B0S3 S1I248 A0A0J6D7Q1 A0A017I312 UPI0003910F49 H9UZ11 E0J3Y2 UPI000512EA8D V1U5Z5 A0A080ECD1 UPI00057C0D33 C8UJJ5 A0A037Y8I6 UPI0002CB816A E8EQ65 L5GW49 A0A0G3PID9 A0A0A8UGD6 A0A0E2U8R4 UPI0006815C5F E8GKX8 S1P4I2 A0A0J4WXG0 UPI0005B345AD E8Y8R2 A0A0H7LQT5 E7Y7G4 V2T0S1 V3D6C7 I6FW96 A0A0E0U5P0 UPI0002CA127F E7WDV6 A0A073UI66 A0A060UYE6 UPI0004713F51 B7L973 UPI0003BB4FC5 E8H1P2 V6FB56 M7P8V6 UPI0002CC83F4 E3PP00 UPI0002C92D2D S4M012 J7RN24 W8XG71 A0A0F5SGW9 A0A0E0Y7I2 A0A0F0YW97 E8CHG5 S0XLH4 V3KJ79 UPI00069BE650 A0A0E3H4E0 A0A0F6K2Y9 S4JDI0 A0A064T2Q3 W8XNG3 A0A069X2G5 C8TYP3 UPI0002515E81 V2N400 M9F528 A0A098GXV9 A0A080FIP4 A0A0E1M3W0 UPI000699EF6E S4LF58 S1D3C3 UPI0004D54D12 A0A073T7U4 A0A090L9E8 V3IA60 E8DQ33 H4KPG7 UPI0005EDDA48 A0A0F1AYX8 A0A0A0F8P2 A0A0I2HXR0 V2P0K5 V8KDE4 UPI0005F08B1C A0A0J1YCS9 UPI0005E69EA7 UPI000579D3C9 A0A0H5PMN6 A0A070P4C7 UPI0002CC5FF6 A0A0J0HLB6 W1G679 A0A0J9AH48 V2ISV7 U9Y365 R0D8R6 A0A0F0RX59 C0VZH1 T9ARP5 V1SA88 V0YN02 UPI0002CB96B1 UPI0005CAF560 UPI0006978729 U9ZZ52 T2PQM4 V4B7K3 A0A0H0HV04 A0A0D7LBX3 UPI0003EE8CC5 B7NFB5 E8AAS2 E9YLR4 S3IGV1 H4JFN3 W1WHJ6 UPI000445D59E E8CTA8 M8LCT6 UPI00068E1050 A0A0J1M123 D8ASL4 UPI0003EF87D0 E7WWF9 M9GJI0 A0A0J4LFX4 X7HIN0 UPI0006695A36 A0A0F4BA88 E8FMH0 L3Q9J9 A0A0H0CH29 A0A064CY91 UPI0002C95A23 V5KL37 E7WRB0 S0X3S7 A0A0J0K9Y5 S0TTK4 W1XFI9 V2MBS7 E7X696 V0Y7G3 W7NZ36 UPI0006520C97 UPI00050ADC02 A0A0H3T6D2 E8GBI9 A0A070PK74 B5EZS8 UPI0002CC3250 W1WI72 X5GT01 V1K5C8 H4L599 H7EDN3 UPI0002CB3E81 UPI0005097CC3 UPI00056EBE4B X0NNF5 S1EDJ9 UPI0002E3BEE2 UPI0004DA7107 Q8KI59 J1QP03 V2AKC0 T9IU72 UPI0004E2422C Q05311 UPI00044FBFBE A0A0J2C8A9 E7VT49 A0A079Y2R2 A0A0J5MX43 A0A0D6IPI8 UPI0005CCFA15 E1ITF3 A0A0J6JML4 A0A0G3J263 A0A085HQF9 A0A0E8MI42 Q9R2U0 A0A0D1CQK0 V1I8L5 H3KWA8 A0A078LAH7 A0A021WR03 UPI00050A604C V6FP78 E8E0U7 H4LJP0 A0A0H0R1B4 S5IH33 V0V674 V2ASN4 S4JVA6 T6GSY1 UPI0005575061 V2KFI5 A0A0A7A0U6 UPI000627EB24 E7ZFI6 T6LNG9 UPI0004D8B75C A0A0H3SHZ2 E2X518 UPI000237C903 V2H9R2 H4IZK5 UPI000452C3C5 E8XJD9 Q329Y9 A0A0J5L635 E8BMX2 T5NEX8 R8WLR8 A0A0H3NUG9 A0A0J1JGH9 A0A085GMJ6 G5NKF6 N3MX37 S0XDX3 V7QPA0 W1FYY2 UPI0005E94CC5 V2A9V9 A0A029P4R5 A0A0A1B385 V1H945 A0A0A1R5N6 U1VBA4 A0A0G2MMZ1 A0A027YRP2 UPI00016C8460 A0A0F6B9B3 A0A073VBC0 UPI00066656EE V1MAM1 L3PWK5 UPI000675DF85 A0A0F7JES7 I6FY95 A0A0H3FP62 B3YFM1 S1GVU2 A0A0E1LGB9 UPI0005F937F6 A0A0D7LIV8 UPI0005014921 A0A0H2WUN6 M9K6A8 D2ZMD4 L0MA89 X7I032 UPI00063C446F V1XNT7 T9TBM0 UPI0003ED146B UPI0004B98CEC D4BE43 UPI0002CAC6D5 A0A0H3S2Q8 D7ZK11 A0A0E2A5Z6 E1I441 UPI0001C3403D UPI00026947D6 X2KCL1 L3NT10 A0A0I2G829 D8ADU8 G5P1B6 UPI0002B60DFC A0A0H3IIW8 H4K9Z1 UPI0002C8DC1E UPI0001FB4B2C G5LGM2 I6FIC1 C0Q3C2 S0VUG2 UPI00066659C9 D7XDB2 A0A0H2VE91 A0A073VVJ1 V2NKZ3 M2P544 UPI0003EF3546 UPI00050B0CB8 Q1R4C1 UPI0003F93F50 A0A0H4VNJ1 E3XW04 UPI000370A2F0 A0A0I0YDW9 W8ZQE8 L4IV51 V7UEH9 S0V315 A0A0J0PQF7 UPI0003FF3A54 A0A024KJK2 A0A0J8YSU2 M4LQ08 N2JTA5 E6WHH6 UPI00067E3DB0 UPI00050B495A UPI0002CAC228 Q57HQ6 H4IIM4 UPI00057BE5A7 UPI00050ABBE7 A0A090ND62 A0A0F0R0L1 A0A089GCQ8 A0A026RVL8 UPI0003BF7FA1 UPI00050BC0F6 A0A024L7U7 U2MK71 S5HQI6 E9XUJ2 T8XXA5 UPI0001FB4D65 I0VX51 UPI000575034C A0A0H3BQS9 A0A017JGC0 A0A0H0BBN2 A0A0I2EFX3 C9XT80 UPI000282E630 V1SQB1 D6JHC8 A0A0F3XJB2 V6E727 UPI0003027365 UPI0005307602 A0A0H3RDJ9 T5ZU25 A0A085PA08 V0VKJ6 K8BR96 UPI0006A629C6 V0GAX0 Q8X8P5 D6IG48 UPI000589632A W0AUM0 UPI0006A6039B A0A0F0IT73 B1LLY4 L4UZM9 UPI0002A4D3B7 UPI0002B9DE03 UPI0002CC68E2 A0A0D5WNL4 R6TVJ8 L3C1J2 UPI000628182D K8A0N1 UPI0002CCA014 A0A0F2ZMT8 A0A0G3JMG2 UPI0004D7F7DF UPI00062757E2 UPI0005196C1F A0A0D5WY30 A0A0G2NZ21 D3QXA5 UPI0006800C6C UPI00053A6F37 E3G3X3 A0A0K0HFZ6 V5ZRD0 D3H4V1 UPI0002CCBDA0 UPI0005CEF8A7 A0A0B5INH2 UPI00056ED442 V7IJT2 A0A023Z641 UPI0002CB0C5E A0A0H7L7Z4 UPI0006969E0D UPI0002CA6A43 V2D935 C6EG01 A0A0J1LKQ0 A0A0I1QVM4 A0A0J8ZBK9 F1ZPQ8 B5Q5I2 B7UND0 A0A0G2NT28 UPI0004643C70 UPI0002EE2722 E9Z1A2 V1RGT6 L9HYA4 V2PRV4 A0A0I2RPB4 A0A0J1RJH0 I2REU8 X4BR52 A0A0H3PUC7 UPI0002CBC0D7 UPI000281D683 A0A066P4B2 UPI0005EA4E43 UPI0004A8DEFA A0A0F6GUU7 B3HAV2 A0A0I0V6U1 F5S3C5 UPI0003BCDF55 G5P176 Q3YVF3 UPI00050A9E00 W1WLH4 A0A0E2M6M6 UPI0002C935D9 G9WCL2 A0A0F6FES0 UPI0004D4FB82 UPI00069A9A9D A0A0J0P9D3 A0A090V7I4 G5MAR0 A0A0G3KBA3 M8PMP0 A0A0H7RCS5 A0A0J0VSA8 A0A089Q428 UPI00067C89D7 A0A0F6CBC7 UPI000483DDB5 UPI00050B3740 A0A0J0QVP3 UPI00039807B5 UPI00067AC747 J2YWY3 A0A0D7ESI8 W1ASV1 A0A0J0LCW8 UPI0004DA8D8C UPI00069F6BC0 B7LU43 A0A0J0DP92 W1DW14 A0A0A6EFN1 UPI0004635F02 UPI0002A6DF22 A0A025C616 UPI000352C78C J2X0N7 A0A0F1A8N0 UPI000463708C UPI00067D0E8D A0A0H4S4M4 G4C8R9 G5LGM3 A0A0F1HGJ1 R9VNE2 UPI00028DE27E A0A0H2Z4N7 UPI0005AA8C72 UPI0002B9DB1F A0A074TPI3 UPI0002CC3EDA UPI0005F857AB F0JWA1 UPI0002CC9A6F X3YLW0 A0A0J9AGF8 UPI0002695288 UPI0005797D3A UPI0005EAF698 UPI0005C674E6 UPI0004381BCD UPI000668E496 UPI00034D611A J5W6W9 D7ZU66 UPI0006658EEE UPI0002AEB5B0 A0A0J1SRM4 M7RF80 A0A0F1L5B3 UPI000696EBE1 A0A0J4TS24 G5QS93 A0A0H0ABS0 K8AAR4 UPI0006675A7B UPI0006995D61 A0A0C7L099 G5MAU7 A0A0J2H3P7 A0A0I2D6J9 E1J5X4 UPI00053B46E6 Z5CP12 X3UNX0 A0A0E2R9B6 M8KEA1 E6BNN4 UPI000681EBB3 D3RH84 B3PGX1 UPI0004B58C5B UPI000574FBCF D7XMB5 UPI0002C925C5 B5XYK3 UPI0002DB7E81 A0A0J2FBS7 UPI0002A1343F A0A079F6E9 B1ERG0 UPI0005CC1957 UPI0003B61D19 A0A0J9AI33 UPI000537C7CA A0A071AVK4 UPI0002CB7FF2 UPI000666ABBF UPI00037AE6F5 A0A0J8Z8W7 A0A0J8Y5W3 A0A079FJR3 A0A029K3W3 R5WI88 UPI00040A8AC5 H3MDK3 UPI000472771C UPI0005AB1B13 A0A029LTL0 A0A089PHR7 A1SQW8 A0A0G3PTS1 UPI0002B580C6 UPI0002CBB03B UPI000390DC2A A0A0H3CV27 G5QNH0 A0A0I1EU55 E8DEL4 UPI00069C71E8 S5N2R7 A0A0H0C242 G5S3E4 UPI000669A104 E7XIB6 X5MS66 G2S5G8 A0A0F0TB45 G5SJ34 W0BDW4 UPI0003915F4D UPI000614634C A0A0F2AUK2 A0A071M1C1 X3XE62 G8LKV0 UPI00038FA10B UPI0005ED8E6D A0A0J0JZA2 UPI00035E9F50 UPI000689139D A0A0J0TK85 A0A0F6TXR6 V1GX81 UPI0006769073 F4W269 G5R9I0 A0A0J0GZG0 UPI00037F6D42 A0A0G3QEA8 A0A090U681 UPI00038FA53E G5P3F2 A0A0G4BNQ9 UPI0006145584 UPI000666A5AA A0A023V4X1 A0A0B7GI73 UPI0003D2FA70 A0A0I0T9Z3 UPI0004DAE8E7 UPI000315529E A0A0I2BUS0 A0A0G2MHY8 A0A084CN62 R4Y7F0 A0A0J8XHN7 A9MJ02 R8WJE0 A0A0H0DHS6 UPI00068E1512 C8T0H7 UPI0003BECD47 S1HNI3 A0A0J0IRI6 A0A0J0SSF2 UPI0005D093A3 F4VLD8 H3N5H9 UPI0003BB87D8 A0A0F3YGX7 A0A0J0B472 A0A0H4R3L7 F4SRL8 K6KT52 UPI000353E7DB UPI0002CB932D A0A0J0ENJ8 A0A0B8UZ32 F4NQE6 A0A0H3ENI0 A0A0F5B4P9 S0UJP4 A0A0H0DM28 A0A0B8V3X1 K8B2N0 UPI0004D4C5A1 UPI000250C01F M8X9A7 A0A0J0PB20 U4TEK6 UPI0003A800E6 UPI000598DBB2 N4NWV1

TABLE 8 D. radiodurans UvrD and its Orthologs in Thermophilic Species Protein Accession # Entry name names Organism Gene name Q9RTI9 Q9RTI9_DEIRA DNA Deinococcus radiodurans (strain DR_1775 helicase ATCC 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/ NCIMB 9279/R1/VKM B-1422) FORMJ1 FORMJ1_DEIPM DNA Deinococcus proteolyticus (strain Deipr_0885 helicase ATCC 35074/DSM 20540/JCM 6276/ NBRC 101906/NCIMB 13154/ VKM Ac-1939/CCM 2703/MRP) H8GTP8 H8GTP8_DEIGI DNA Deinococcus gobiensis (strain DSM uvrD2, helicase 21396/JCM 16679/CGMCC 1.7299/ DGo_CA1449 I-0) C1CVA3 C1CVA3_DEIDV DNA Deinococcus deserti (strain VCD115/ uvrD, helicase DSM 17065/LMG 22923) Deide_12100 A0A016QL30 A0A016QL30_9DEIO DNA Deinococcus phoenicis DEIPH_ctg079orf0093 helicase Q1J014 Q1J014_DEIGD DNA Deinococcus geothermalis (strain Dgeo_0868 helicase DSM 11300) D3PR99 D3PR99_MEIRD DNA Meiothermus ruber (strain ATCC K649_05745 helicase 35948/DSM 1279/VKM B-1258/ 21) (Thermus ruber) A0A0D0N7B7 A0A0D0N7B7_MEIRU DNA Meiothermus ruber SY28_04645 helicase E8U932 E8U932_DEIML DNA Deinococcus maricopensis (strain Deima_1926 helicase DSM 21211/LMG 22137/NRRL B- 23946/LB-34) D7BGJ6 D7BGJ6_MEISD DNA Meiothermus silvanus (strain ATCC Mesil_1937 helicase 700542/DSM 9946/VI-R2) (Thermus silvanus) A0A0A7KLI4 A0A0A7KLI4_9DEIO DNA Deinococcus swuensis QR90_10300 helicase F2NK78 F2NK78_MARHT DNA Marinithermus hydrothermalis Marky_1312 helicase (strain DSM 14884/JCM 11576/T1) A0A0F7JIM6 A0A0F7JIM6_9DEIO DNA ‘Deinococcus soli’ Cha et al. 2014 SY84_01165 helicase E4U8J8 E4U8J8_OCEP5 DNA Oceanithermus profundus (strain Ocepr_1221 helicase DSM 14977/NBRC 100410/VKM B- 2274/506) L0A7L7 L0A7L7_DEIPD DNA Deinococcus peraridilitoris (strain Deipe_3622 helicase DSM 19664/LMG 22246/CIP 109416/KR-200)

TABLE 9 36 seed sequences of UvrD-like helicase group PF00580 ADDA_BACSU EX5B_MYCTU O53348_MYCTU PCRA_GEOSE Q9ZJE1_HELPJ UVRD_ECOLI ADDA_LACLM HMI1_YEAST O66983_AQUAE PCRA_MYCTU REP_BUCAP UVRD_HAEIN EX5B_BORBU O24736_THETH O83140_TREPA PCRA_STAA8 REP_ECOLI UVRD_MYCGE EX5B_CHLTR O25569_HELPY O83991_TREPA Q46538_DICNO REP_HAEIN UVRD_MYCPN EX5B_ECOLI O26611_METTH O84614_CHLTR Q9Z7D4_CHLPN SRS2_SCHPO UVRD_RICPR EX5B_HAEIN O51319_BORBU P73465_SYNY3 Q9ZCJ7_RICPR SRS2_YEAST Y340_MYCPN

TABLE 10 Selected Low-Cysteine or No-Cysteine Wild-Type PcrA Helicases PcrA with no cysteine from L. citreum MK20 /gene=″pcrA″ MSVETLTNGMNNKQAEAVQTTEGPLLIMAGAGSGKTR /locus_tag=″LCK_00476″ VLTHRIAHLVQDLNVFPWRILAITFTNKAAREMRERIAA /EC_number=″3.6.1.-″ LLSEDVARDIWVSTFHALAVRILRRDGEAIGLAKNFTIID /note=″COG0210L; TSAQRTLMKRVINDLNLDTNQYDPRTILGMISNAKNDM TIGR01073″ LRPRDYAKAADNAFQETVAEVYTAYQAELKRSQSVDF /codon_start=1 DDLIMLTIDLFQSAPEVLARYQQQFEYLHVDEYQDTND /transl_table=11 AQYTIVNLLAQRSKNLAVVDGAGQSIYGWRGANMNNI /product=″ATP-dependent LNFEKDYPNAHTVMLEQNYRSTQNILDAANAVINHNNE DNA helicase PcrA″ RVPKKLWTENGKGDQITYYRAQTEHDEANFILSNIQQLR /protein_id=″ACA82309.1″ ETKHMAYSDFAVLYRTNAQSRNIEESLVKANMPYSMV /dh_xref=″GI:169803691″ GGHKFYERKEILDIMAYMSLITNPDDNAAFERVVNEPKR (SEQ ID NO: 53) KFLTFAEMMHNLRQQSEFLNVTELTELVMTQSGYRQM LAEKNDPDSQARLENLEEFLSVTKEFDDKYQPEDPESIDP VTDFLGTTALMSDLDDFEEGDGAVTLMTLHAAKGLEFP VVFLIGLEEGIFPLSRAMMDEDLLEEERRLAYVGITRAM KKLFLTNAFSRLLYGRTQANEPSRFIAEISPELLETAYSGL SRDKTQKKTLPFDRKMQRATATTYQATPVTKITNGVTG GDQTSWSTGDKVSHKKWGVGTVISVSGRADDQELKVA FPSEGVKQLLAAFAPIQKVD Selected Low Cysteine count thermophilic PcrA helicases >tr|B5Y6N2|B5Y6N2_COPPD MALPQENLIPPSPSHNHLTLSLRSHIGGFFIYNEDVDSVDL DNA helicase SKLNEAQKQAVTAPPKPLAIIAGPGSGKTRVLTYRALFA OS = Coprothermobacter VKEWHLPPERILAITFTNKAADELKERLGRLIPEDGRIFA proteolyticus (strain ATCC 35245/ ATMHSFAARMLRYFAPYAGISQNFVIYDDDDSKGLIEDI DSM 5265/BT) GN = pcrA LKQMNMDTKRFRPNDVLNHISAAKARMFDCNTFPEFIR PE = 4 SV = 1 (SEQ ID NO: 54) QRYGSWGYYFDTVHQVFMTYERLKEQSQALDFDDLIM VLAQRMEDRPELREMIAGLFDLVMVDEFQDTNFAQYQ MLLYMTNPHYSGMNNVTIVGDPDQSIYGFRAAEYYNIK FRIDDYNPEVVFLDLNYRSNRTIVSDASALINDSPSALFE RKLESIKGAGNKLILRRPFDDADAAITAAFEVQRLHKMG IPYEEIAVLMRTRALTARVEREFATRNIQYHIIGGVFPFFAR REIKDILAYLRLSRNAMDRVSLKRILTMKKRGFGTASLE KLFNFAEENKLTLLEAMKAAVESLLFKKLSMNDYLESL YTLIQTIQEIAEPSQAIYLVMEQENLLDHFRSISKSEEEYIE RTENVKQLISIAEESADMDDFLQRSALGTRENNGGVEGV AISTVHGVKGLEFQAVILYYVTDGFFPHSLSVTTAEKEEE RRLLYVAMTRAKEHLIFYVPYKQPWGNGFEQMARPSPF LRSIPKELWDGKPNEIESLYAPYSPQQKWSE >tr|E8MZN5|E8MZN5_ANATU MDSLEHLNPQQRAAVTASAGPVLVLAGPGSGKTRVLTF DNA helicase OS = Anaerolinea RIGYLLSQLGVAPHHILAVTFTNKAAREMQSRVEKLLGH thermophila (strain DSM 14523/ SLQGMWLGTFHAICARILRREQQYLPLDANFVIFDEDDQ JCM 11388/NBRC 100420/ QALIKRALRDLNLDEKLYRPTSVHAAISNAKNNLILPED UNI-1) GN = pcrA PE = 4 SV = 1 YPTATYRDEVVARVYKRYQELLVSSNAVDFDDLLLYA (SEQ ID NO: 55) WKLLNEFSTVREQYARRFEHILVDEFQDTNLAQYELVK LLASYHRNLFVVGDEDQSIYRWRGADYRNVLRFEEDFP DRQKILLEQNYRSTQRVLDAAQAVINRNRNRTPKRLKST PEHGEGEKLVLYEAVDDYGEAAFVVDTIQQLVAGGKA RPGDFAIMYRTNAQSRLLEEAFLRAGVPYRLVGAMRFY GRREVKDMIAYLRLVQNPADEASLGRVINVPPRGIGDKS QLALQMEAQRTGRSAGLILMELGREGKDSPHWQALGR NASLLADFGSLLGEWHRLKDEISLPSLFQRILNDLAYREY IDDNTEEGQSRWENVQELLRIAYEEKGLTAFLENLAL VSDQDTLPENVEAPTLLTLHAAKGLEFPIVFITGLDDGLIP HNRSLDDPEAMAEERRLFYVGLTRAKKRVYLVRAAQR STYGSFQDSIPSRFLKDIPADLIQQDGRGRRMGRSWQSES RRSWDDNYAGTWGSRPERAKPSHAPILQPRFKPGMRVK HPSWGEGLVVDSRIQDEDETVDIFFDSVGFKRVIASIANL EILS >tr|E8PM35|E8PM35_THESS MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGA DNA helicase  GSGKTRTVVHRVAYLIAKRGVFPSEILAVTFTNKAAEEM OS = Thermusscotoductus  RERLKRMVKGGGELWVSTFHSAALRILRVYGERVGLKP (strain ATCC 700910/ GFVVYDEDDQTALIKEVLKELGLAARPGPLKALLDRAK SA-01) GN = pcrA1 PE = 4 SV = 1 NRGEAPESLLSELPDYYAGLSRGRLLDVLKRYEEALKA (SEQ ID NO: 56) QGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEY QDTNPVQYRFTKLLAGEEANLMAVGDPDQGIYSFRAAD IKNILEFTRDFPGAKVYRLEENYRSTEAILRFANALIVNN ALRLEKTLRPVKPGGEPVRLYRARDARDEARFVAEEILR LGPPFDRVAVLYRTNAQSRLLEQTLASRGVPARVVGGV GFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPPRGIG PATVEKVEALAREKGLPLFEALRVAAEVLPRPALRHFL ALMEELQELAFGPAEGFFRHLLEATDYPAYLREAYPED YEDRLENVEELLRAAKEAEGLMEFLDKVALTARAEEPG EPAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHRSSL STLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRTE ATRPSRFLEEVEGGLYEEYDPYRASAKVSPSAPGEARA SKPGAYRGGEKVIHPRFGQGTVVAAMGDEVTVHFEGV GLKRLSLKYADLRPVG >tr|E8PL08|E8PL08_THESS MLNPEQEAVANHFTGPALVIAGPGSGKTRTVVHRIARLI DNA helicase RKGVDPETVTAVTFTKKAAGEMRERLVHLVGEETATK OS = Thermusscotoductus  VFTATFHSLAYHVLKDTGTVRVLPAEQARKLIGEILEDL (strain ATCC 700910/ QAPKKLTAKVAQGAFSRVKNSGGGRRELIALYTDFSPYI SA-01) GN = pcrA2 PE = 4 SV = 1 ERAQDAYEAYKEEKRLLDFDDLLHQAVHELSTDIDLQA (SEQ ID NO: 57) RWGHRARFLIVDEYQDTNLVQFNLLRLLLTPEENLMAV GDPNQAIYAWRGADFRLILEFKKHFPNATVYKLHTNYR SHNGIVTAAKKVITHNTQREDLDLKALRNGDLPTLVQA QSREDEALAVAEVVKRHLDQGTPPEEIAILLRSLAYSRPI EATLRRYRIPYTIVGGLSFWNRKEVQLYLHLLQAASGNP ESTVEVLASLVPGMGPKKARKALETGKYPKEAEEALQL LQDLRAYTGETGEHLASAVQNTLHRHRKTLWPYLLELA DGIEEAAWDRWANLEEAVSTLFAFAHHTPEGDLDTYLA DILLQEEDPEDSGDGVKIMTLHASKGLEFAVVLLPFLVE GAFPSWRSAQNPATLEEERRLFYVGLTRAKEHAYLSYH LVGERGATSPSRFARETPANLIHYNPTIGYQGKETDTLSK LAELF

Example 10 Cysteine Reactive Crosslinkers and Alternative Crosslinkers

Bis-maleimide crosslinkers with contour length varying from 6 to 25 Angstrom were used as exemplary crosslinkers (Table 2): BMPEG2, BMOE, BMH, DTME, (1,2-Phenylene-bis-maleimide), and (Succinyl Bis[(phenylimino)-2,1-ethanediyl]bis(3-maleimidopropanamide)). Alternatively bis-maleimide crosslinkers such as BMPEG3, BMB, BMDB, (1,4-Phenylene-bis-maleimide), (Bis-maleimidomethyl), and (N,N-[Dithiobis[(carbonylphenylimido)-2,1-ethanediyl]]bis(3-maleimidopropanamide)) or homobifunctional vinylsulfone crosslinker such as HBVS can be used. An alternative crosslinker can be of any crosslinker of desired length that fits the criteria set forth in Example 8 with suitable functional end groups. For crosslinking two cysteines, suitable end groups can be any of the maleimide, haloacetyl, iodoacetyl, pyridyl disulfide, vinylsulfone and other suitable moieties. Table 11 shows examples of bis-maleimide linkers with corresponding lengths.

TABLE 11 Selected Bismaleimide Crosslinkers Spacer Arm Composition Crosslinker Spacer Arm Length (Å) (between maleimide groups) BMOE 8.0 Alkane BMDB 10.2 Cis-diol (periodate cleavable) BMB 10.9 Alkane BMH 13.0 Alkane DTME 13.3 Disulfide (reducing agent cleavable) BM(PEG)2 14.7 Polyethylene glycol (PEG) BM(PEG)3 17.8 Polyethylene glycol (PEG)

Example 11 Alternative Crosslinking Methods to Cysteine Crosslinking

As an alternative to cysteine crosslinking chemistry, one can introduce a pair of unnatural amino acids for crosslinking with linkers using different chemistries as defined herein. This may be advantageous over cysteine engineering, because it may eliminate the extra steps of site directed mutagenesis of potentially interfering native cysteines and potentially detrimental effects of such mutations in other related helicases. For example, it was shown herein that in the PcrA helicase, there are two native cysteines that are highly conserved across diverse species (FIGS. 4A and 4B). The mutating out of these two cysteines in PcrA from Bacillus stearothermophilus reduced the ATPase activity by more than 80%. However replacing all five native cysteines in Rep from E. coli had a very minimal effect.

Alternatively, a target residue pair can be introduced, one of which is an unnatural amino acid and the other is a cysteine. Alternatively, one can introduce two or more pairs of target residues, preferably each pair can be specifically targeted with specific crosslinkers that employ orthogonal chemistries so that unwanted inter-pair crosslinking is avoided (for example, one pair of cysteines and one pair of unnatural amino acid residues) for enhanced conformational stability and activity.

Example 12 Unnatural Amino Acids as an Alternative to Cysteine Crosslinking

There are nearly one hundred unnatural amino acids (Uaa) that have been genetically incorporated into recombinant or endogenous proteins. These Uaa provide a wide spectrum of side chains that can be covalently crosslinked using a homo or hetero bi-functional linker with suitable end groups. Additionally a multi-branched multi- or homo-functional crosslinkers can be used for secondary conjugation other chemicals, biomolecules such as a DNA polymerase enzyme, in addition to the main crosslinking reaction. Uaa can incorporate specific reactive groups to the specific sites on the proteins, such as aryl iodides, boronic acids, alkynes, azides, or others, or they can be post-transcriptionally or chemically modified to prepare for desired crosslinking chemistry. Examples of Uaa include, but are not limited to, homopropargylglycine, homoallylglycine, azido-phenylalanine, azidohomoalanine and others. Uaa modification and crosslinking reactions include, but are not limited to, azides and cyclooctynes in copper-free click chemistry, nitrones and cyclooctynes, oxime/hydrazone formation from aldehydes and ketones, tetrazine ligation, isonitrile based click reaction, quaricyclane ligations, copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition, copper acetylide to activate terminal alkynes toward reaction with azides, Staudinger ligation, cyclooctyne reactions, and Huisgen cycloaddition. Suitable end groups of these crosslinkers would include, but are not limited to, azide, alkyne, succinimide, phosphine, etc.

Example 13 Selected Super-Family 1B (SF1B) and Super-Family 2 (SF2) Helicases

Selected SF1B and SF2 helicases are described herein. In an embodiment, the helicase is RecD2. In an embodiment, the RecD2 helicase is from D. radiodurans. Selected target residue pairs for crosslinking and the specific distances between the pairs, in RecD2 are shown in FIG. 12 and Table 12.

TABLE 12 Selected Crosslinking Pairs for 5′ to 3′ SF1B Superhelicase RecD2 Backbone C—C RecD2 distance in Å ALA632 ILE170 18.0 ALA632 ASN171 17.0 PHE635 GLY200 18.0 1B domain amino acid 2B domain amino acid Backbone C—C (RecD2; D. radiodurans) (RecD2; D. radiodurans) distance in Å ARG 410 (B-sheet) ASN 596 (loop) 12.91 PRO 413 (B-sheet) PHE 603 (loop) 13.04 GLN 414 (B-sheet) ASN 596 (loop) 11.13 GLY 415 (loop) GLU 601 (loop) 8.38 PHE 416 (loop) ARG 417 (loop) 6.36 ARG 417 (loop) ASN 599 (loop) 12.43 GLY 418 TYR 598 (loop) 11.00 LEU 411 (B-sheet) PHE 603 (loop) 13.62 ARG 417 (loop) ARG 417 (loop) 10.14

RecQ helicase has a winged helix domain (denoted by WH, shown in green in FIG. 13 and FIG. 14) that rotates 90 degrees and makes contact with the duplex in the unwinding conformation (Mathei et al., “Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases” Proc Natl Acad Sci USA. 2015 Apr. 7; 112(14):4292-7). In an embodiment, stabilization of the WH domain of RecQ leads to superhelicase activation. Stabilization of the closed form of the WH domain can be achieved by crosslinking it to the catalytic core using the residue pairs shown in Table 13.

TABLE 13 Selected Crosslinking Pairs for Superhelicase RecQ Backbone C—C Catalytic domain WH domain distance in Å PHE221 VAL470 7.91 GLU219 ARG514 5.61 LYS212 GLU467 8.90 PHE221 GLU467 6.52

RecQ1 helicase also has a winged helix domain (denoted by WH, shown in green in FIG. 15) that rotates 90 degrees and makes contact with the duplex in the unwinding conformation. In an embodiment, stabilization of the WH domain of RecQ1 leads to superhelicase activation. Stabilization of the closed form of the WH domain can be achieved by crosslinking it to the catalytic core using the residue pairs shown in Table 14.

TABLE 14 Selected Crosslinking Pairs for Superhelicase RecQ1 Zinc finger alpha WH beta hairpin Backbone C—C helix domain amino acid domain amino acid distance in Å MET429 TYR564 12.17 VAL431 THR566 8.31 MET429 ALA565 8.77 MET429 THR566 7.10

5′-3′ SF1 superhelicase T4 Dda (FIG. 16) is known to unwind dsDNA as a monomer, and has sequence similarity to E. coli recD (exonuclease V). In an embodiment, stabilization of the tower/hook and pin domains leads to superhelicase activation. Stabilization of the closed form of the tower/hook and pin domains can be achieved by crosslinking them using the residue pairs shown in Table 15. Wild-type T4 Dda has 439 amino acids, a 5′-3′ unwinding polarity, and 5 cysteines. It is a DNA helicase that stimulates DNA replication and recombination reactions in vitro, and has been suggested to play a role in the initiation of T4 DNA replication in vivo. It acts by dissociating and associating with the DNA molecule being unwound, interacting with UvsX and binding tightly to the gene 32 protein. Selected crosslinking pairs that parallel SF1A helicases are located in the tower/hook and the pin domains based on the crystal structure (FIG. 16) and are listed in Table 15.

TABLE 15 Selected Crosslinking Pairs for Superhelicase T4 Dda 1B domain (pin) 2B domain (tower/hook) Backbone C—C amino acid amino acid distance in Å THR 91 (B-sheet) TRP 374 (Alpha helix) 9.77 TYR 92 (B-sheet) TYR 363 (Alpha helix) 11.78 TYR 92 (B-sheet) TYR 363 (Alpha helix) 11.73 TYR 92 (B-sheet) LYS 364 (Alpha helix) 10.42 GLU 93 (loop) LYS 364 (Alpha helix) 6.83 GLU 93 (loop) ALA 372 (loop) 9.25 GLU 93 (loop) PRO 373 (loop) 10.45 GLU 93 (loop) SER 375 (Alpha helix) 10.38 GLU 94 (loop) TRP 374 (Alpha helix) 8.25 GLU 94 (loop) ALA 372 (Alpha helix) 8.25 GLU 94 (loop) SER 375 (Alpha helix) 10.73 GLU 94 (loop) TRP 378 (Alpha helix) 8.58 VAL 96 (B-sheet) LYS 381 (Alpha helix) 12.55 VAL 96 (B-sheet) TRP 374 (Alpha helix) 12.36 VAL 96 (B-sheet) TRP 378 (Alpha helix) 10.56

Structural data have been Obtained for the SF1B RNA helicase Upf1 (5′-3′ SF1B RNA/DNA helicase) in complexes with phosphate, ADP and the non-hydrolysable ATP analogue, ADPNP (Cheng et al, 2006), although a structure with bound RNA remains lacking. These structures reveal a conformational change that accompanies binding of ATP and which is very similar to that which occurs during catalysis in SF1A helicases such as PcrA.

Example 14 Identifying Suitable Crosslinking Sites for Immobilizing 2B Domain at a Particular Rotational Conformation between the Open and Closed Form

It has been shown herein that the closed and open forms captured in the crystal structures are the active and the inactive states of the Rep helicase, respectively, which can be interconverted by a 133 degree rotation of the 2B domain around an axis. Therefore, the active conformation can be defined through definition of the range of a rotational angle, θ (theta), relative to the closed form with θ=0 (FIG. 17). For example, in an embodiment, Rep-X becomes a superhelicase if θ<40 degrees. In addition, arresting the helicase in an intermediate conformation, such as, e.g. θ=40 degrees, may allow a new function. While immobilizing the 2B domain at an angle θ=40 degrees, it was found that residue pairs distances increase more than 10 Å when θ changes from 40 degrees to 0 degrees (to closed form), and increase more than 20 Å when θ changes from 40 degrees to 130 degrees (to open form). Positions of residues at the desired θ, can be interpolated from open and closed form crystal structures via rigid body rotation of the 2B domain around an axis. Having performed this calculation for θ=40 degrees of Rep helicase, it was found that 2B residues that satisfy this criteria are residues 515 and 518-525, and the residues on the rest of the protein structure satisfying the criteria are residues 543-547. For example, crosslinking residues 521 to residue 547 on with a crosslinker with a length of about 10 Å, restricts the 2B domain to a conformation of θ=40 degrees. Similar to restricting the 2B conformation to θ=0 degrees (closed form), corresponding residues to restrict in helicases with unknown structures can be determined via sequence alignment.

Rigid body rotation of the 2B domain around a chosen axis can convert the closed form to the open form or vice versa. In the case of E. coli Rep, the chosen axis intersects the alpha carbons of residue ILE371 and residue SER280 or residue ALA603. In an embodiment, the chosen axis intersects the alpha carbons of residue ILE371 and residue SER280. Theta is the angle of rotation around this chosen axis from the closed form toward the open form. According to this definition, theta is 0 degrees for the closed form. In the case of E. coli Rep, theta increases to 133 degrees when it is rotated around the chosen axis to obtain the open form. Theta for the open form may vary between different helicases.

Thus, in an embodiment of a modified helicase described herein, the first amino acid and second amino acid, together with an axis vector defined by an alpha carbon of ILE371, from which the vector originates, and an alpha carbon of SER280 or an alpha carbon of ALA603 of E. coli Rep helicase, define an angle, theta, wherein theta is about 355 degrees to about 25 degrees in an active conformation. In an embodiment, theta is about 355 degrees, about 0 degrees, about 5 degrees, about 10 degrees, about 15 degrees, about 20 degrees or about 25 degrees, or any increment or point between about 355 degrees to about 25 degrees. In another embodiment, theta is about 0 degrees in an active conformation. In an embodiment, theta is about 60 degrees to about 155 degrees in an inactive conformation. In an embodiment, theta is about 60 degrees, about 65 degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85 degrees, about 90 degrees, about 95 degrees, about 100 degrees, about 105 degrees, about 110 degrees, about 115 degrees, about 120 degrees, about 125 degrees, about 130 degrees, about 133 degrees, about 135 degrees, about 140 degrees, about 145 degrees, about 150 degrees, or about 155 degrees, or any increment or point between about 60 degrees to about 155 degrees. In another embodiment, theta is about 133 degrees in an inactive conformation. In an embodiment, the axis vector is defined by an alpha carbon of ILE371 and an alpha carbon of SER280 of E. coli Rep helicase. In another embodiment, the axis vector is defined by an alpha carbon of ILE 371 and an alpha carbon of SER280 of E. coli Rep helicase.

Example 15 Examples of Thermophilic Orthologs/Homologs of UvrD, Rep and PcrA

Based on the crosslinking target site selection criteria established in Example 8, and analogous to identification of suitable crosslinking sites in hologous helicases as described in Example 9, by sequence alignment and structural homology modeling, the corresponding crosslinking target residues are identified in helicases with unknown structures. Subsequently these helicases can be converted to superhelicase forms. Thus, in an embodiment, Rep-like thermophilic helicases featuring low or no cysteine content, and homologs or orthologs thereof are also suitable candidates for cross-linking to form a thermophilic superhelicase. Selected examples of thermophilic orthologs or homologs of UvrD, Rep and PcrA are shown in Tables 16-18. In certain exemplary embodiments, a suitable UvrD, Rep or PcrA helicase is selected from the following species: Thermococcus sp. EXT9, Thermococcus sp. IRI48, Thermococcus sp. IRI33, Thermococcus sp. AMT7, Thermococcus nautili, Thermococcus onnurineus (strain NA1), Thermococcus kodakarensis (strain ATCC BAA-918/JCM 12380/KOD1) (Pyrococcus kodakaraensis (strain KOD1)), Thermococcus sibiricus (strain MM 739/DSM 12597), Thermococcus paralvinellae, Thermus aquaticus Y51MC23, Thermus aquaticus Y51MC23, Thermus aquaticus Y51MC23, Thermus sp. RL, Thermus sp. RL, Thermus sp. 2.9, Salinisphaera hydrothermalis C41B8, Thermus filiformis, Meiothermus ruber, Thermus sp. NMX2.A1, Thermus thermophilus JL-18, Thermus scotoductus (strain ATCC 700910/SA-01), Thermus scotoductus (strain ATCC 700910/SA-01), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Thermus oshimai JL-2, Thermus oshimai JL-2, Thermus oshimai JL-2, Thermomonospora curvata (strain ATCC 19995/DSM 43183/JCM 3096/NUMB 10081), Thermodesulfatator indicus (strain DSM 15286/JCM 11887/CIR29812), Geobacillus stearothermophilus (Bacillus stearothermophilus), Coprothermobacter proteolyticus (strain ATCC 35245/DSM 5265/BT), Meiothermus silvanus (strain ATCC 700542/DSM 9946/VI-R2) (Thermus silvanus), Anaerolinea thermophila (strain DSM 14523/JCM 11388/NBRC 100420/UNI-1), Thermoanaerobacterium thermosaccharolyticum M0795, Meiothermus ruber (strain ATCC 35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Meiothermus ruber (strain ATCC 35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Deinococcus radiodurans (strain ATCC 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/NCIMB 9279/R1/VKMB-1422), Thermodesulfobium narugense DSM 14796, Thermus thermophilus (strain HB8/ATCC 27634/DSM 579), Dictyoglomus thermophilum (strain ATCC 35947/DSM 3960/H-6-42), Thermus thermophilus (strain SG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermus sp. CCB_US3_UF1, Deinococcus geothermalis (strain DSM 11300), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM 7039), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM 7039), Marinithermus hydrothermalis (strain DSM 14884/JCM 11576/T1).

TABLE 16 Gene names Protein (primary)/Gene Entry (3D) Entry name names Organism Length encoded by L0B9N8 L0B9N8_9EURY UvrD Rep Thermococcus 591 Plasmid helicase SFI sp. EXT9 pEXT9a L0B9J0 L0B9J0_9EURY UvrD Rep Thermococcus 547 Plasmid helicase SFI sp. IRI48 pIRI48 L0BAD9 L0BAD9_9EURY UvrD Rep Thermococcus 591 Plasmid helicase SFI sp. IRI33 pIRI33 L0BAT5 L0BAT5_9EURY UvrD Rep Thermococcus 591 Plasmid helicase sp. AMT7 pAMT7 W8NUG2 W8NUG2_9EURY Superfamily I Thermococcus 665 DNA and nautili RNA helicase and helicase subunits B6YXQ7 B6YXQ7_THEON UvrD/REP Thermococcus 533 helicase onnurineus (strain NA1) Q5JFK3 Q5JFK3_THEKO DNA Thermococcus 661 helicase, kodakarensis UvrD/REP (strain ATCC BAA- family 918/JCM 12380/ KOD1) (Pyrococcus kodakaraensis (strain KOD1)) C6A075 C6A075_THESM DNA Thermococcus 716 helicase, sibiricus (strain UvrD/REP MM 739/DSM family 12597) W0I5I1 W0I5I1_9EURY DNA Thermococcus 659 helicase, paralvinellae UvrD/REP family protein B7AA42 B7AA42_THEAQ DNA helicase Thermus 701 (EC 3.6.4.12) aquaticus Y51MC23 B7A5I6 B7A5I6_THEAQ DNA helicase Thermus 868 (EC 3.6.4.12) aquaticus Y51MC23 B7A954 B7A954_THEAQ DNA helicase Thermus 542 (EC 3.6.4.12) aquaticus Y51MC23 H7GEQ7 H7GEQ7_9DEIN DNA helicase Thermus sp. RL 1030 (EC 3.6.4.12) H7GH69 H7GH69_9DEIN DNA helicase Thermus sp. RL 693 (EC 3.6.4.12) A0A0B0SAG4 A0A0B0SAG4_9DEIN DNA helicase Thermus sp. 2.9 692 (EC 3.6.4.12) A0A084IL47 A0A084IL47_9GAMM ATP- Salinisphaera 670 rep dependent hydrothermalis DNA helicase C41B8 Rep (EC 3.6.4.12) A0A0A2WMV1 A0A0A2WMV1_THEFI DNA helicase Thermus 665 (EC 3.6.4.12) filiformis A0A0D0N7B7 A0A0D0N7B7_MEIRU DNA helicase Meiothermus 706 (EC 3.6.4.12) ruber W2U4X3 W2U4X3_9DEIN DNA helicase Thermus sp. 710 (EC 3.6.4.12) NMX2.A1 H9ZQB5 H9ZQB5_THETH DNA helicase Thermus 693 (EC 3.6.4.12) thermophilus JL-18 E8PM35 E8PM35_THESS DNA helicase Thermus 708 pcrA1 (EC 3.6.4.12) scotoductus (strain ATCC 700910/SA-01) E8PL08 E8PL08_THESS DNA helicase Thermus 621 pcrA2 (EC 3.6.4.12) scotoductus (strain ATCC 700910/SA-01 E4U8J8 E4U8J8_OCEP5 DNA helicase Oceanithermus 719 (EC 3.6.4.12) profundus (strain DSM 14977/ NBRC 100410/ VKM B-2274/ 506) E4U4N5 E4U4N5_OCEP5 DNA helicase Oceanithermus 917 (EC 3.6.4.12) profundus (strain DSM 14977/ NBRC 100410/ VKM B-2274/ 506) E4UAI1 E4UAI1_OCEP5 DNA helicase Oceanithermus 889 Plasmid (EC 3.6.4.12) profundus (strain pOCEPR01 DSM 14977/ NBRC 100410/ VKM B-2274/ 506) E4UAI8 E4UAI8_OCEP5 DNA helicase Oceanithermus 638 Plasmid (EC 3.6.4.12) profundus (strain pOCEPR01 DSM 14977/ NBRC 100410/ VKM B-2274/ 506) E4UAI4 E4UAI4_OCEP5 AAA ATPase Oceanithermus 606 Plasmid profundus (strain pOCEPR01 DSM 14977/ NBRC 100410/ VKM B-2274/ 506) K7QW32 K7QW32_THEOS DNA helicase Thermus oshimai 693 (EC 3.6.4.12) JL-2 K7QWX5 K7QWX5_THEOS DNA helicase Thermus oshimai 694 Plasmid (EC 3.6.4.12) JL-2 pTHEOS01 K7QTS9 K7QTS9_THEOS DNA helicase Thermus oshimai 854 (EC 3.6.4.12) JL-2 D1AF88 D1AF88_THECD DNA helicase Thermomonospora 799 (EC 3.6.4.12) curvata (strain ATCC 19995/ DSM 43183/ JCM 3096/ NCIMB 10081) F8A884 F8A884_THEID DNA helicase Thermodesulfatator 503 (EC 3.6.4.12) indicus (strain DSM 15286/ JCM 11887/ CIR29812) A0A087LEB0 A0A087LEB0_GEOSE Uncharacterized Geobacillus 807 protein stearothermophilus (Bacillus stearothermophilus) B5Y6N2 B5Y6N2_COPPD DNA helicase Coprothermobacter 696 pcrA (EC 3.6.4.12) proteolyticus (strain ATCC 35245/DSM 5265/BT) D7BJL0 D7BJL0_MEISD DNA helicase Meiothermus 646 Plasmid (EC 3.6.4.12) silvanus (strain pMESIL02 ATCC 700542/ DSM 9946/VI- R2) (Thermus silvanus) E8MZN5 E8MZN5_ANATU DNA helicase Anaerolinea 737 pcrA (EC 3.6.4.12) thermophila (strain DSM 14523/JCM 11388/NBRC 100420/UNI-1) L0INW7 L0INW7_THETR ATP- Thermoanaerobacterium 769 Plasmid dependent thermosaccharolyticum pTHETHE01 exoDNAse M0795 (Exonuclease V), alpha subunit/ helicase superfamily I member D3PR99 D3PR99_MEIRD DNA helicase Meiothermus 706 (EC 3.6.4.12) ruber (strain ATCC 35948/ DSM 1279/VKM B-1258/21) (Thermus ruber) D3PLL2 D3PLL2_MEIRD DNA helicase Meiothermus 920 (EC 3.6.4.12) ruber (strain ATCC 35948/ DSM 1279/VKM B-1258/21) (Thermus ruber) Q9RTI9 Q9RTI9_DEIRA DNA helicase Deinococcus 745 (X-ray (EC 3.6.4.12) radiodurans crystallog- (strain ATCC raphy (3)) 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/NCIMB 9279/R1/VKM B-1422) M1E5C5 M1E5C5_9FIRM DNA helicase Thermodesulfobium 610 (EC 3.6.4.12) narugense DSM 14796 Q5SIE7 Q5SIE7_THET8 DNA helicase Thermus 692 (EC 3.6.4.12) thermophilus (strain HB8/ ATCC 27634/ DSM 579) B5YD55 B5YD55_DICT6 DNA helicase Dictyoglomus 656 (EC 3.6.4.12) thermophilum (strain ATCC 35947/DSM 3960/H-6-12) F6DJA4 F6DJA4_THETG DNA helicase Thermus 722 Plasmid (EC 3.6.4.12) thermophilus pTHTHE1601 (strain SG0.5JP17-16) F6DIL2 F6DIL2_THETG DNA helicase Thermus 692 (EC 3.6.4.12) thermophilus (strain SG0.5JP17-16) F6DJ67 F6DJ67_THETG DNA helicase Thermus 1014 Plasmid (EC 3.6.4.12) thermophilus pTHTHE1601 (strain SG0.5JP17-16) G8N9P8 G8N9P8_9DEIN DNA helicase Thermus sp. 704 (EC 3.6.4.12) CCB_US3_UF1 Q1J014 Q1J014_DEIGD DNA helicase Deinococcus 741 (EC 3.6.4.12) geothermalis (strain DSM 11300) Q745W4 Q745W4_THET2 DNA helicase Thermus 551 Plasmid (EC 3.6.4.12) thermophilus pTT27 (strain HB27/ ATCC BAA-163/ DSM 7039) Q72IS0 Q72IS0_THET2 DNA helicase Thermus 692 uvrD (EC 3.6.4.12) thermophilus (strain HB27/ ATCC BAA-163/ DSM 7039) F2NK78 F2NK78_MARHT DNA helicase Marinithermus 716 (EC 3.6.4.12) hydrothermalis (strain DSM 14884/JCM 11576/T1)

TABLE 17  SEQ ID  Entry Sequence NO:  L0B9N8 MSEALPVTSFEFSLPEESVIKIYGPPGTGKTTTLVRIIEHLIGEHDHTEFLESYGLSLLFGQYGAEDV 58 IFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAIDGVITQNFGSLSPEDW FRLFCRQNGLRFESSEMGYSNVFNDGNRLWNALTWAYNVYYPTKGPKARHEALKRLAPKL WKYPPLWEEYKTEKGILDYNDMLVKAYEGLKSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLQFE IFRLLANYMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVLKRSYRLPIVVQAKAMTVISKTR HRKEKTVAPRTDLGDFKYKLFWFPDFLNDLVREAQEGHSIFILVRTNRQVLKLGKELILAGVHF RHLKVDYRSIWEAGSKEWGTFRDLVQALLKARRGEELEIADLVTILYYSELIDWHLGEKLPEKER YKKIAEQMEKTIEAIEKGLMPFDILKVKDDPFSVLDLEKIESLSPRHGKVAVELIREIMKEKSQVV SVPRDAEIYLDTLHASKGREADVVFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKKVYLL NGKHPFPVL L0B9J0 MRVKIYGPPGTGKTTTLQRTIDYTLGNSSEPPIPLPESFPTDLEPKNLAFVSFTNTAIDVIGKRTGI 59 TTRSKEAPYMRTIHGLILSVLAEHFDPVAVDNLGKLADIQAEFSMRMGYYYSKDPFEFAEGN MKFNVITRALELYLPKTGDVEEALKLIDNREDRKFALAWYRYKRQKKIMDFDDILVIGYEHLEDF YVPVEVAFIDEGQDNGPLDYILLEKGFEGAKFVFLAGDPLQSIYGFKGADPRLFVRWKADKEIIL PRSYRLPKKVWLLSQSWALSLGIKGAVVRYAPSEKLGRVSRMKFIEALSYAVEQAKRGRSVLIL ARTNSLVKFVGNILSIEFGVAYGHLKRASYWESHLLKFIEGLQMLKLWDGVFPIKVQDTKPITGL IRKLKDKHAREVLRRWRDSRQWSLEVQAVLQRIKKNPSEYFYITDFDRQALKAYFSKARLDLTE ELIIDTIHAAKGEEADVVIFLDFIPTRSEERINPEELQEKLVAYVGFTRAREELIIVPTPAIKYHPMR DFMGVRQILGVVNFHKHLLIKELVGGL L0BAD9 MSEALPVTSFEFSLPRERIIKLYGAPGTGKTTTLVKIIEHLIGFQDHTEFLENYGINLPFGQYEPGE 60 VIFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAVDGLITQNFGSLSPED WFRNFCRQNGLRFESSEMGYSNVFNEGNQLWNALTWAYNVYYPTKGPKARYEALKRLAPK LWKFPPLWEEYKKGRGILDYNDMLVRAYEGLRSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLQ FEIFRLANHMDLVIIAGDDDQTIFSYQGADPRLMNYVPGLEWLRKSHRLPIVVQAKALTVISK TRHRKEKTVAPRTDLGDFKYKLFWFPDFLNDLVREAQEGHSIFILVRTNRQVLKLGKELILAGV HFEHLKVDYRSIWEAGSKEWGTFRDLVQALLKAKRGEELEVADLVTILYYSELIDWHLGEGISE KERYKKIAEQMEKTIEAIEKGLMPFDVLRVKENPFSVLDLEKIESLSPRHGKVAVELIKELMKEKS QVVSIPKDARIYLDTLHASKGREADVVFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKK VYLLNGKHPFPVL L0BAT5 MSEALSITSFDFTLPRERIIKIYGPPGTGKTTTLVRIIEHLIGFQDHTEFLENYGLSLPFGQYGAEDV 61 IFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAVDGLITQNFGSLSPED WFRHFCRQNGLRFESSEMGYSNIFNEGNQLWNALTWAYNVYYPTKGPKARYEALKRLAPKL WKFPPLWEEYKKEKGILDYNDMLIRAYEGLKSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLQFEI FRLLANHMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVLSKSYRLPIVVQAKALTVISKTRH RKEKTVAPRTDLGDFKYKLFWFPDFLNDLVREAQEGHSIFILVRTNRQVLKLGKELILAGVHFEH LKVDYRSIWEAGSKEWGTERDLVQALLKAKKGEELEVADLVTILYYSELIDWHLGERISEKERYK KIAEQMEKTIEAIEKGLMPFDILKVKENPFSVLDLEKIESLSPRHGKVAVELIKELMKEKSQVVSIP KDAKIYLDTLHASKGREADVVFLINDLPRKWSNILKTREELDAERRVWYVGLTRARKKVYLLNG KHPFPIL W8NUG2 MNENEKLSKFIAKLKVLIEMERKAEIEAMRAEMRRLSGREREKVGRAVLGLNGKVIGEELGYFL 62 VRYGREREIKTEISVGDLVVISKRDPLKSDLVGTVVEKGKRFITVALETVPEWALKSVRIDLYAND ITFKRWLENLENLRESGRRALELYLGLREPEGGEEVEFTPFDKSLNASQRRAIAKALGSPDFFLIH GPFGTGKTRTLVELIRQEVARGNRVLATAESNVAVDNLVERLVDSGLKVVRVGHPSRVSRGLH ETTLAYLMTQHELYGELRELRVIGENLKEKRDTFTKPAPKYRRGLTDRQILRLAEKGIGTRGVPA RLIREMAQWLKINEQVQKTFDDARKLEERIAREIIREADVVLTTNSSAGLEVVDYGSYDVAIIDE ATQATIPSVLIPINRAGREVLAGDHKQLPPTILSEKAKELSKTLFEGLIERYPGKSEMLTVQYRMN ERLMEFPSREFYDGRIEADESIRRITLADLGVKSPEDGDAWAEVLKPENVLVFIDTARREDRFER QRYGSESRENPLEARLVKEAVEGLLRLGVKAEWIGVITPYDDQRDLISSLLPEEIEVKTVDGYQG REKEVIVLSFVRSNRKGELGFLKDLRRLNVSLTRAKRKLILIGDSSTLSSHPTYRRLVEFVRERETV VDAKRLIGKVKIK B6YXQ7 MTAPIPTTYSILGVAGAGKTTQLIDLLNYLNFENSRNEKIWERHFEPVELNRIAFISFSNTAIQEIA 63 NRTGIEIKARKKSAPGRYFRTVTGLAEVLLYENNLMTFEEVRSVSKLEGFRIKWAREHGMYYKP RDNDISYSGNEFFAEYSRLVNTYYHVKSLSEIIEMHSKSHLLLDYIREKEKLGIVDYEDILMRAYDY RNDIVVDLEYMIIDEAQDNSLLDYATLLPIAKNNATELVLAGDDAQLIYDFRGANYKLEHKLIER SEIILNLTETRRFGSEIANLATAIIDDMNYIQKREVLSAATHSTKVAHIDLFQMMSILQNMATTD LTVYILARTNAVLNYVAKVLDEYKIQYKKNERITDFDRFLLSLNRLMRNEYTNDDIYTIYNYLRNK VAREEELKERLFQHKLHWTEKDVLGILLLAYEQTTAKRILTTAKNTNFKIKLSTIHSAKGSEADVV ELINSVPHKTKMKILENYEGEKRVLYVAVTRARKFLFIVDQPVARRYEQLYYIRSYESRAQGSLV NRVAVPVA Q5JFK3 MNEKEVLLSKFIAHLKELVEMERRAEIEAMRLEMRRLSGREREKVGRAVLGLNGKVIGEELGYF 64 LVRYGRDREIKTEISVGDLVVISKRDPLKSDLVGTVVEKGKRFLTVAIETVPEWALKGVRIDLYAN DITFKRWMENLDNLRESGRKALELYLGLREPEESEPVEFQPFDKSLNASQRGAIAKALGSGDFF LVHGPFGTGKTRTLVELIRQEVARGHKVLATAESNVAVDNIVERLADSGLKVVRIGHPSRVSKA LHETTLAYLITQHDLYAELRELRVIGENLKEKRDTFTKPAPKYRRGLSDREILRLAEKGIGTRGVPA RLIREMAEWIRINQQVQKTFDDARKLEERIAREIIQEADVVLTTNASAGLEVVDYGEYDVAVID  EATQATIPSVLIPINRAKREVLAGDHKQLPPTILSEKAKELSKTLFEGLIERYPEKSEMLIVQYRM NERLMEFPSREFYDGKIKAHESVKNITLADLGVSEPEFGNFWDEALKPENVLVFIDTSKREDRF ERQRRGSDSRENPLEAKLVTETVEKLLEMGVKPDWIGVITPYDDQRDLISSMVGEDIEVKTVD GYQGREKEIIVLSFVRSNRRGELGFLTDLRRLNVSLTRAKRKLIAVGDSSTLSNHPTYRRFIEFVRE RGTFIEIDGKKH C6A075 MTRVQIPAGAPKYGPVAQPGQSARLISGRSGVRSPAGPPKALLKERFRELFIHKNPVITMHVK 65 NYIAKLVDLVELEREAEIEAMREEMRRLKGYEREKVGRAILNLNGKIIGEEFGFKLVKYGRKEAF KTEIGVGDLVVISKGNPLASDLVGTVVEKGSRFIVVALETVPSWAFRNVRIDLYANDITFRRQLE NLKKLSESGIRALKLILGKETPLKSSPEEFTPFDRNLNQSQKEAVSYALGSEDFFLIHGPFGTGKTR TLVELIVQEVKRGNKILATAESNVAVDNLVERLWGKVKLVRLGHPSRVSVHLKESTLAFQVESH ERYRKVRELRNKAERLAVMRDQYKKPTPQMRRGLTNNQILKLAYRGRGSRGVPAKDIKQMA QWITLNEQIQKLYKFAEKIESEIIQEIIEDVDVVLSTNSSAALEFIKDAEFDVAIIDEASQATIPSVLIP IAKARREVLAGDHKQLPPTILSEEARALSETLFEKLIELYPFKAKMLEIQYRMNQLLMEFPSREFY NGKIKADGSVKDITLADLKVREPFFGEPWDSILKREEPLIFVDTSNRTDKWERQRKGSTSRENP LEALLVREIVERLLRMGIKKEWIGIITPYDDQVDSIRSIIQDDEIEIHTVDGYQGREKEIIILSLVRSN KKGELGFLMDLRRLNVSITRAKRKLVVIGDSETLVNHEYKRLIHFVKKYGRYIELGDTGIN W01511 MNLIRYINHLKELVELEREAEIEAMREEMRKLTGYEREKVGRAVLGLNGKIIGEEFGYKLVKYGR 66 KQEIKTEISVGDLVVISKGNPLASDLIGTVTEKGKRFLVVALETVPSWALRNVRIDLYANDITFKR QIENLDKLSESGKRALRFILGLEKPKESIDIEFKPFDEQLNESQKKAVGLALGSEDFFLIHGPFGTG KTRTVAEVILQEVKRGKKVLATAESNVAVDNLVERLWGKVKLVRLGHPSRVSKHLKESTLAYQ VEIHEKYKRVREFRNKAERLAMLRDQYTKPTPQWRRGLTDRQILRLAEKGIGARGIPARVIKS MAQWITFNEKVQRLYNEAKKIEEEIVKEIIRQADVVLSTNSSAALEFIKDIKFIDVAVIDEASQATI PSVLIPIAKANKFILAGDHKQLPPTILSEEAKELSETLFEKLIELYPSKAKMLEIQYRMNERLMEFPS KEFYNGKIKAYDGVKNITLLDLGVRVFSFGEPWDSILNLKEPLVFVDTSKHPEKWERQRKGSLS RENLLEAELVKEIVQKLLRMGIKPESIGVITPYDDQRDLISLLLENDEIEVKTVDGYQGREKEVIILS FVRSNKKGELGFLTDLRRLNVSLTRAKRKLIAIGDSETLSAHPIYKRFVEFVKEKGIFVQLNQYVS QTS B7AA42 MGEAHPSEEALLSSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIARRGVFPSEIL 67 AVTFTNKAAEEMKARLKAMVRGAGELWVSTFHAAALRILRVYGERVGLKPGFVVYDEDDQT ALLKEVLKELGLAAKPGPIKSLLDRAKNQGVPPEHLLLELPEFYAGLSRGRLQDVLHRYQEALRA QGALDFGDILLYALKLLEEDGEVLKRVRKRARFIHVDEYQDTNPVQYRFTRLLAGEEANLMAV GDPDQGIYSFRAADIRNILDFTQDYPKARVYRLEDNYRSTEAILRFANAVIVKNALRLEKTLRPV KKGGEPVRLFRAESARDEAREVAEEIARLGPPFDRVAVLYRTNAQSRLLEQALASRGIPARVVG GVGFFERAEVKDLLAYARLSLNPLDAVSLKRVLNTPPRGIGPATVEKVQAIARERGLPLFEALKV AALTLPRPEPLRAFLALMEELMDLAFGPAEAFFRHLLLATDYPAYLKEAYPEDAEDRLENVEELL RAAKEAESLMDFLDKVALTARAEEPAEAEGRVALMTLHNAKGLEFPVVFLVGVEEGLPHRSS LSTQEGLEEERRLFYVGVTRAQERLYLSYAQEREIYGRLEPVRPSRFLEEVDEGLYEVYDPYRQSS RKPTPPPHRALPGAFRGGEKWEIPRFGPGTVVAAAGDEVTVHFEGVGLKRLSLKYADLRPA B7A5I6 MRVYLASAGTGKTHALVEELKGLIQSGVPLRRIAALTFTRKAAEELRGRAKRAVLALSAEDPRLK 68 EAEREVHGALFTTIHGEMAEALRFITAPLLSLDPDFALLDTFLAEALFLEEARSLLYRKGLDGGLA RALLHLYRKRTLAETLHPLPGAEGVFALYLEALEGYRRRLPAELSPSDLEALALRILENPEALRRVV ERFPHILLDEYQDTGPLQGRFFQGLKEAGARLVWGDPKQSIYLERNARVEVEREALKQAEEVR YLSTTYRHAQAVAEFLNRFTALFGEEGVRVRPHRQEVGRVEVHWVVGEGGLEEKRRAEAHLL LDRLMALREEGYAFSQMAVLVRSRSSLPPLEAAFRARGVPYALGRGRSFFARPEVRDLYHALR LSLLEGPPGPEERLALLAFLRGPWVGLDLSEVEEALKAQDPIPLLPEAVRAKLRALRALAGLPPLE ALKRLSRDEAFLRRLSPRARVNLDALLLLAAMERFPDLEALLEWLRLRAEDPEAAELPEGEEGV QVLTVHGAKGLEWPWALFDLSRGENPKEEDLLVGLGGEVALRGTPAYKEVRKALRKAQAEE ARRLLYVALSRARDVLIVTGSASGRPGPWVEALERLGLGPESQDPLVRRHPFKALPPLGDRPQ TPPPPPLPAPYAHLAFPERPLPFVYSPSAFTKAKEPVPLAEALEKEALPEFYRALGTLVHYAIARH LDPEDEGAMAGLLLQEVAFPFAEGEKRRLLEEVRDLLRRYRGMLGPSLPPLEAREEDHAELPLV LPLGGTVWYGILDRLYRVGGRWYLEDYKTDREVRPEAYREQLAIYRRALLEAWGVEAEARLVY LRHGLVHPLDPEELERALKEGFPGMGPGEGGEKA B7A954 MKGLTGSSRLRVYGPPGTGKTTWLKNEVERLLRSGVPGEEIAVCAFSRAAFREFASRLAGQVP 69 EENLGTIHSLAYRAIGRPPLALTKDALSDWNRRVPDTWRVTPRVDGRGADLLDVMDPYEDE DSRPPGDKLYDRVAYLRNTLAPMAAWSEEERAFFQAWKSWMNAKGLVDEPGMLEAALAK PGGLGARFLLVDEAQDLTPLQLLLVEKWAQGARLALVGDDDQAIYGFMGADGASFLGVPVE DELVLGQSYRVPARVQRVAEAVIRRVQNRAPKRYAPRGDEGEVRLLWVPPEDPYHAWDAL ERVNRGESVLFLATAKYLLEELKRELLRVGEPYANPYAPHRHSFNLFPQGARSAWEKARSFLFP NRIAADVKAWTKHVSSKVFAVKGEEARRYIESFPDEEKVGDDHPIWNVFRPEHRPHAVGRD VSWLLDHLLGNAPKTMRQSLMVALKSPEAVLQGRARVWIGTIHSVKGGEADWVYVWPGY TRKAAREHPDQLHRLFYVAATRARKGLVLMDQGKAPHGYVWPRVDEFWGEVWV H7GEQ7 MEANLYVAGAGTGKTYTLAERYLGFLEEGLSPLQVVAVTFTERAALELRHRVRQMVGERSLG 70 HKERVLAELEAAPIGTLHALAARVCREFPEEAGVPADFQVMEDLEAALLLEAWLEEALLEALQ DPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRXLRRRMEELFHG ERPEERYPGFPKGWRXEEPEVVPDLLAWAGEVKFNKKPWLEYKXDPALXRLLKLLGGVKEGES PGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRALEXEEVRAYYRGRERR LLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQSIYSFRRADPKVMERFQXEAAKEGLRV RRLEKSHRYHQGLADFHNRFFPPLLPGYGAVSAERKPEGEGPWVFHFQGDLEAQARFIAQEV GRLLSEGFQWDLGEKAYRPMSLRDVAVLGRTWRDLARVAEALRRLEVPAVEAGGGNLLETR AFKDAYLALRFLGDPXDEEALVGLLRSPFFALTDGEVRRLAEARGEGETLWEVLEREGDLSAEA ERARETLRGLLRRKALEAPSRLLQRLDGATGYTGVAARLPQGRRRVKDWEGTLDLVRKLEVGS EDPFLVARHLRLLLRSGLSVERPPLEAGEAVTLLTVHGAKGLEWPVVEVLNVGGWNRLGSWK NNKTKPLFRPGLALVPPVLDEXGNPSALFHLAKRRVEEEEKQEENRLLYVAATRASERLYLLLSP DLSPDKGDLDPQTLIGAGSLEKGLEATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPLAAR DPEAARRRLLGEPEXEGGEAWXPXXPQETEEEVPGGAGVGRMTHALLERFEAXEDLEREGRA FLEESFPGAEGEEVEEALRLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVGED WVLDYKTDRGVDAXAYLLQVGVYALALGKPRALVADLREGKLYEGASQQVEEKAEEVLRRLM GGEGQGRQPYPLAATDPGHGAPG H7GH69 MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVTFT 71 NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEV LKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALD FGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQG IYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRXANAVIVKNALRLEKALRPVKRGGEPV RLYRAEDAREEAREVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARVVGGVGFFE RAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAART FXRAEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRLENVEELLRAAK EAEDLQDFLDRVALTAKAEEPAEAEGKVALMTLHNAKGLEFPVVELVGVEEGLLPHRNSLSTLE GLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRXPKPXPPP HRPRPGAFRGGERVVHPREGPGTVVAAQGDEVTVHFEGXGLKRLSLKYAELXPA A0A0B0SAG4 MDEALLSSLNEAQRQAVLHFQGPALVVAGAGSGKTRTVVHRVAYLIAHRGVYPTEILAVTFTN 72 KAAEEMRERLKGMVRGAGEVWVSTFHAAALRILRVYGERVGLKPGFVVYDEDDQTALLKEV LKELGLSAKPGPIKALLDRAKNRGEPPEALLAELPEYYAGLSRRRLLDVFFRYQEALKAQGALDF GDILLYALRLLEEDQEVLARVRKRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMAVGDPDQG IYSFRAADIKNILQFTADFPGAKVYRLEENYRSTEAILRFANAVIVKNALRLEKTLRPVKRGGEPV RLFRAKDAREEARFVAEEILRLGPPFDRIAVLYRTNAQSRLLEQALAGRGVGARVVGGVGFFER AEVKDLLAYARLALNPLDSVSLKRILNTPPRGIGPAIVEKVARLAQEKGLPLFEALKRAELLPRPE PVRHFVALMEELMDLAFGPAEAFFRHLLQATDYPAYLREAYPEDHEDRLENVEELLRAAKEAE SLLDFLDKVALTARAEEPAGAEGKVELMTLHNAKGLEFPWELVGVEEGLLPHRNSLNTLEALE EERRLFYVGVTRAQERLYLSYAEEREVYGRLEATRPSRFLEEVEEGLYQEYDPYRSPRPVPPSHR PKPGAFKGGEKVVHPREGPGTVVAASGDEVTVHFEGVGLKRLSLKYADLRPA A0A084IL47 MALPKLNPQQDAAMRYLDGPLLVLAGAGSGKTGVITRKIAHLIARGYDARRVVAVTFTNKAA 73 REMKQRASKLISADDARGLTVSTFHSLGLQMIREEHAALGYKPRFSIFDSEDADKVLADLVGR DGDHRKATKAAISNWKSALIDPETATAQATGSDIPLARAYGEYQRRLKAYNAVDFDDLLALPV HLLSTDHEARERWQSRFRYLLVDEYQDTNAAQYEMMRLLAGARAAFTVVGDDDQSIYAWR GARPGNIADLSRDFPHLKVIKLEQNYRSVGNVLSAANQLIGASNQRAYEKTLWSAMGPGDRV RVIAAPDEAGEAERIASEISSHKLRLGTAYGDYAILYRGNFQSRAFEKALRERDIPYRVSGGRSFF ERSEIRDLVTYLKLMVNPDDDAAFLRIVNLPRREIGPATLEALGRYAGSRHISLFDAARGIGLAG GVGERSGRRLADFVDWLRNLTQDSEGMTPRELVSQLIVDIDYRNWLRDTSANTKAARKRIEN LDDFIGWLDRLDNAEDGKPVTLEDVVRRLSLMDFANQSEKDVENQVHLLTLHAAKGLEFDH VFLAGLEEGMLPHHACLEDDKIEEERRLLYVGITRARKTLALTYARKRRRGGEESDSVPSRFLEEL PADELDWPSATGTRSKAANAEQGRDQVAALRAMLGASADS A0A0A2WMV1 MPQVGFTDHFFKGLEALSREEQNRVREAVFAFMQDPKHPSFKLHRLEDIKTDRFWSARVSK 74 DLRLILYHHPEMGWIFAYVGHHDDAYRWAETHQAEVHPKLGLLQIFRWEEVRVEPRKIKPLL PDYPDDYLLDLGVPPSYLKPLRLVETEDQLLGLIEGLPQDVQERLLDIAAGRPVTLPPKLAPSEE WFKHPLSRQHIHFIQNLDELRQALSYPWERWMVFLHPAQREAVERVFQGPARVTGPAGTG KTVVALHRAAALARRYPEEPLLLTTFNRFLASRLRSGLQRLLGEVPPNLTVENLHSLARRLHEQH VGPVKLVKEEDYGPWLLEAAQGLEYGKNFLLSEFAFADAWGLYTWEAYRGFPRTGRGVPLTA RERLKLFGAFQKVWGRMENEGALTENGLLHRLRQRAEEGALPRFRAVVVDEAQDLGPAELLL VRALAQEAPDSLFFALDPAQRIYKSPLSWQALGLEVRGRSIRLKVNYRTTREIAKRAEAVLPKEV  EGEMREVLSLLQGPEPEIRGFPTQEACQAELVRWLRWLLEQGVRPEEVAVLARVRKLAEGLA EGLRRAGIPVVLLSDQEDPGEGVRLGTVHSAKGLEFRAVALFGANRGLFPLESLLREAPSEADR EALLAQERNLLYVAMSRARERWVGYWDEGSPFLTP A0A0D0N7B7 MSDLLSSLNPSQREAVLHFEGPALVVAGAGSGKTRTVVHRIAYLLRERRVYPAEILAVTFTNKA  75 AGEMKERLEKMVGRSARDLWVTTFHAAAVRILRTYGEYVGLKPGFVIYDEDDQNTLLKEVLK  ELELEAKPGPFRSMIDRIKNRGAGLAEYMREAPDFIGGVPRDVAAEVYRRYQNSLRMQGALD  FNDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVQYRLTRLLAGERPNLMVVGDPDQ  SIYGFRNADINNILDFTKDYPGARVIRLEENYRSSSSILRVANAVIEKNALRLEKVLRPTKPGGEPV  RLYRAPNAREEAAFVAREIVKLGGYQQVAVLYRTNAQSRLLEEHLRRANVPVRLVGAVGFFER  REIKDLLAYGRVAVNPDDSINLRRIVNTPPRGIGATTVARLVEHAQKTGITVFEAFRAAEQVISR  PQQVQAFVRLLDELMEAAFESGPTAFFQRVLEQTGFREALKQEPDGEDRLQNVEELLRAAQD  WEEEEGGSLADDFLDSVALTAKAEEPQGGDAPVEATLMTLHNAKGLEFPTVFLVGLEENLLPHR NSLHRLEDLEEERRLFYVGITRAQERLYLSYAEERETYGKREYTRPSRFLQDIPQDLLKEVGAFGD  GETRVLSQARPEPKPRTQPAEFKGGEKVKHPKFGSGTVVAAMGGEVTVMFPGVGLKRLAVK  FAGLERLE W2U4X3 MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIAKRGVFPS  76 EILAVTFTNKAALEMRERLKRMVKGAGELWVSTFHSAALRILRVYGERVGLKPGFVVYDEDD  QTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRLLDVLKRYEEALK AQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMA VGDPDQGIYSFRAADIKNILEFTRDFPGAKVYRLEENYRSTEAILRFANALIVNNALRLEKTLRPV  KPGGEPVRLYRARDARDEARFVAEEILRLGPPFDRVAVLYRTNAQSRLLEQALASRGVPARVV GGVGFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPPRGIGPATVEKVEALAREKGLPLFEALR  VAAEVLPRPAPLRHFLALMEELQELAFGPAEGFFRHLLEATDYPAYLREAYPEDHEDRLENVEE  LLRMKEAEGLMEFLDKVALTARAEEPGEPAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHR  SSLSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRA  SAKVSPSPAPSEARASKPKPGAYRGGEKVIHPRFGQGTVVAAMGDEVTVHFEGVGLKRLSLK  YADLRPVG H9ZQB5 MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLVARRGVEPSEILAVTFT 77 NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEV LKELALSARPGPIKALLDRAKNRGVGLEALLGELPEYYAGLSRGRLADVLVRYQEALKAQGALD FGDILLYALRLLKEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQG IYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRLANAVIVKNALRLEKALRPVKRGGEPV RLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARVVGGVGFFE RAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAART SSRVEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRLENVEELLRAAK EAEDLQDFLDKVALTAKAEEPAEAEGKVALMTLHNAKGLEFPVVFLVGVEEGLLPHRNSLSTLE GLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRVPKPAPPP HRPRPGAFRGGERVVHPRFGPGTVVAAQGDEVTVHFEGFGLKRLSLKYAELRPA E8PM35 MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIAKRGVFPS 78 EILAVTFTNKAAEEMRERLKRMVKGGGELWVSTFHSAALRILRVYGERVGLKPGFVVYDEDD QTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRLLDVLKRYEEALK AQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMA VGDPDQGIYSFRAADIKNILEFTRDFPGAKVYRLEENYRSTEAILRFANALIVNNALRLEKTLRPV KPGGEPVRLYRARDARDEAREVAEEILRLGPPFDRVAVLYRTNAQSRLLEQTLASRGVPARVV GGVGFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPPRGIGPATVEKVEALAREKGLPLFEALR VAAEVLPRPAPLRHFLALMEELQELAFGPAEGFERHLLEATDYPAYLREAYPEDYEDRLENVEE LLRAAKEAEGLMEFLDKVALTARAEEPGEPAGKVALMTLHNAKGLEFPWFWGVEEGLLPHR SSLSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRA SAKVSPSPAPGEARASKPGAYRGGEKVIHPREGQGWVAAMGDEVTVHFEGVGLKRLSLKYA DLRPVG E8PL08 MLNPEQEAVANHFTGPALVIAGPGSGKTRTVVHRIARLIRKGVDPETVTAVTFTKKAAGEMR 79 ERLVHLVGEETATKVETATFHSLAYHVLKDTGTVRVLPAEQARKLIGEILEDLQAPKKLTAKVAQ GAFSRVKNSGGGRRELIALYTDFSPYIERAWDAYEAYKEEKRLLDFDDLLHQAVHELSTDIDLQ ARWQHRARFLIVDEYQDTNLVQFNLLRLLLTPEENLMAVGDPNQAIYAWRGADFRLILEFKK HFPNATVYKLHTNYRSHNGIVTAAKKVITHNTQREDLDLKALRNGDLPTLVQAQSREDEALAV AEVVKRHLDQGTPPEEIAILLRSLAYSRPIEATLRRYRIPYTIVGGLSEWNRKEVQLYLHLLQAAS GNPESTVEVLASLVPGMGPKKARKALETGKYPKEAEEALQLLQDLRAYTGERGEHLASAVQN TLHRHRKTLWPYLLELADGIEEAAWDRWANLEEAVSTLFAFAHHTPEGDLDTYLADILLQEED PEDSGDGVKIMTLHASKGLEFAVVLLPFLVEGAFPSWRSAQNPATLEEERRLFYVGLTRAKEH AYLSYHLVGERGATSPSRFARETPANLIHYNPTIGYQGKETDTLSKLAELF E4U8J8 MSARDLLSSLNEQQQAAVQHFLGPALVIAGAGSGKTRTVVHRVAYLLAEREVYPAEVLAVTFT 80 NKAAGEMRERLSRMVGRAAGELVVVSTFHSASLRILRRYGERIGLKPGFVVYDDDDQRVLLKE VLGSLGLEARPTYVRAVLDRIKNRMWSVDEFLAHADDWVGGLTKQQMAEVYARYQQRLAE NNAVDFNDLLLRTIELFERHPEALEAVRQRARFIHVDEYQDTNPAQYRLTKLLAGDEANLMVV GDPDQSIYGERNADIQNILGFERDYRGAVVYRLEANYRSTAAILRVANALIERNQQRLEKTLRP VKPAGEPVRLYRAPDHREEAAFVAREVARLAGERALDDFAVLYRTNAQSRVLEEAFRRLNLPA RIVGGVGFYERREVKDVLAYARLAVNPADDVALRRVINVPARGVGAASVGKLAAWAQAQG VSLLEAAHRAGELLAARQAAAVAKFTDLLTTLREAAEGTGPEAFLRLVLAETGYSEMLRREGDS EPRLENLEELLRAAAEWEEEHGGSVAEFLDEIALTARAEEPNAAPEKSVTLMTLHNAKGLEFPV VFWGVEEGLLPHRSSLGSDAEIEEERRLLYVGITRAQERLYLTLSEERETWGQRERVRPSRFLEE IPEDFLKPVGPFGDAHEPAPAPLSSAPVNRAAKGSASGRIGGEKVRHPRYGEGTVVATSGEG ARQEVTVHFAEAGLKRLLVKYAGLERIE E4U4N5 MKVRIASAGTGKTYALTSRFTAALAEHPPYRLAAVTFTRSAAAELKARLRERLLAIAAGREQPSG 81 AEDVPPEAVVRRAGALATEVLGATVTTIHGFFAELLRQNALALGLEPDFLRIDASESQQIFAEEA RAYVYLNEEDDALAEVLGRLFAKRSLAAELRPQGEAAEALWAHFRAVLERYARRLGGEALGPA DIELHAWRLLERAGREEALAARIRSRLARVEVDEYQDTSPLQGRVFAALEALGVEVEVVGDPK QSIYAFRNADVEVEREAMRRGEPLPPLVTSWRHDRALVRFLNRYVDWVAEERPEAFARAEA PPVEARPDAGPGRVRLQLVQGEARQDALRPYEADQLARWLQERHAEHAWRDMAVLVRSH SSVPLLVRALAAHGLPHVVVGGRGFYDLIEVRDLVHAARVALDPRGRFSLAAFLRGPFAGLDL GRVERVLAAEDPLAELERAAPEVAERVDRLVRWVQTLRPLDFFERMVRTPFLEGASYLERLEP PARANVDQLLFKLASRRYGRLEFLLRDLEDLRGSDEAGVPEGGFDAVRIYTMHGSKGLEWPV VAVFDLNRGQPDGAEPFYVRPGSGEFAAEGDPDYPRFAAEWKERERQEAYRLLYVALSRPRS RLLLSLSVQLKPDGEGLRPKEWRRTLGRTLIEEMNLAAWDALEVERLDAARLPAPKAAAAAPR RAADVDERLRAPVEPLARPPWSPSALKAERPAPPELDDEGDVAVELEEPGVDPGLVARTVGIL VHYAIGQDWGPERLQDLWNQEAVQRLTEPERTRVKTEVAQRLETYWRLLGTELPALDERDE DYAEFPLLLPTRTARLDIVWEGVIDRLYRVGDVWVLEDYKTDRELHPERYHFQLALYRRAVAA AWGIEPEARLVYLRFGEVVPLEAQLLEEAFERGTREAEEV E4UAI1 MKVIVASAGTGKTTRLTQRYLEHLEQHPPQRVAAVTFTNKAAAELRERIFEALGRGSFYDFTPS 82 PALAERLADYQVRVLEAPIGTIHSFFGYLLRLTAPMLGLDPHFEVIDPATARAWFLEEVRNLAIIE GAEVDETVTTALVELFKRRSISEAFEGTGDASRSLVAGFKKVYARWLTRLGGRYLDPSEIERRAL ALIRHPEALERVRSRLGVVLVDEYQDTAPIQARVFEALEEAGVPIEVVGDPKQSIYAFRDADVE GFREAHRRARENGNVETLTVSYRHPPALADFLNAFTSAEAALGKAFTAEEAPEVKPGREGDAR VELITVTPGDGKATLDALRNGEARLLARELRRLHDEEGYDYGQMLVLFRRRHQLPPLLRALRG AGLPFAVVGLRGLYEEPEVRELYHALRLATGEAPRDSLAVFLSGPFGGLTLGQVREVLAQDAPE SYLTLHHPEAAERLLRLRADAEKMRPAEALTRLIEAPTAKGPPFLDLLELEMADTVLYVLGRIEH TRTYPEAVATLESFRSGGEEEASLARLGGDAVRVMSAHAAKGLQAPVVVIFDADRTFNGNSD ELVIEPRTGRVALNGEDAYESIAQALKARKEGEDHRLIYVALSRSSERLIVSAAVKEPRKGSWLH HLTEVLNLGSKFEHRNVTLAEIALEEPIEQEAATLPVDPELATPLPPAPPAVSSPTALKAERELEV PDPEEAWPADPEARLLGRIVGILVHEGIQRDWDPDDPEVLLALEGEQVLEEVPADRRPAVIEE VATLLRVYRTLLGSAIPSLEEREVDLAELPLVYPLGATAWEGVIDRLYRVGDVWYLEDYKTDREV HPERYHSQLALYREAVRKHWGIEPEVRLWLRTGQVVPLDAAALKEGLASYTGG E4UAI8 MNEHERVIAHEVGPAAVVAGAGSGKTRAATLRAARLARTGERVGLVTFTASAAEEMRQRVL 83 AEDVPAKHVWAGITHSLAFQILRQFPEAGGYEGFPEVLTPNDELRLFRRLWAELLDQDLDAEL RRKLVKALGFFRKARAEEALEGWAARAGESLELDAEMLEALMISFQLRKREAGLASFDDLIEG ASRALGDKDVRKWADRRFPFLIVDEYQDTSRAQETFLAALMPGEAPNLMVIGDPNQAIYGW RGAGSRTFERFQARYPQAVLYPLRKNYRSTRAVLRLAERAIARLYRSGQEAYYRLEGVKEEGEP PVLLTPPNAAAEATDVAREVARAVASGVPPEEIAVLARSSMQLAGVEDRLARLGVATRLLGGI RLSERREVICTLVQLIXAAWSLHERALVDFIEEAVPGLGERTLTRVEHAARPYNLVDRIMNDGA FVRGFSTRVQQGLFMTRTLLQLARATFEGVTGEAFAERFREFAQDLYGELLPGYLARIGKQGP NEEARRRHLEREVATVEAFAREEAEGGLDDLLARLAFLEQQDGPAVTLGTVHAVKGLEFEVVF WGMVEGAFPLADDSDPEEERRLFYVAATRAKRRLYLSAPTYGPRGKILQPSRYLEEALDEGLV RLQKVRPAA E4UAI4 MVSEGRWKIERVVYLKDGFAVVAVRNEAGERHTAVGEMPTPVEGTWVRMETEHTVHPRY 84 GPRLRVVRFLGLAPPPSKELAKIEGYLKLGFSEEAASWIAAREGSRPERAFDKPQELLVPGVPRE VLRRVFPRLERLLGGLIDLLGEGHTAAPLFLLAERSGLGKEEIQELAREARKQRLIVEEQGRYGLV QPYRTERSIADGLLFRLKPGRGLRLTPPAGHGLSDEQARIFKLVRENRVVVLTGGPGSGKTTTIA TLLAAPELHRMREGIAAPTGKAARRIAEVARLPAETIHRLLGLGEARRPLYHARNPLPYDLLVID  ETSMLDAEIAAFLVDALAPSTSVIFVGDPDQLPPVGPGQFLRDLMTRVATLRLTQIFRQAQDS PIVNGAYALREGRMPLADGERLRLLPFEEEAAQTTLRTLLDELQRLEQIVGERPQVLVPGNRGP LGVRRLSPFLQQQLNPGGKPLGPIGWGMEAREGDPAVWIHNDYELGIMNGEVGVLRGGGS LGLTFETPTDRFAIPGNKRSRLVLAYAMTVHRSQGSEWPAVITILPKAHMALLSRELVYTALTRS KQYHTLLFHPEALYRARAVQASRRYTWLDVLLRG K7QW32 MTAPGHPDALLAPLNPAQQEAVLHFQGPALVVAGAGSGKTRTVVHRVAYLMAHRGVYPGE 85 ILAVTFTNKAAEEMKGRLKALVPGAGELWVATFHSAALRILRVYGEAIGLKPGRNYDEADQE ALLKEVLKELGLSAKPGPLKALLDRAKNRGEAWEALEIPDYYAGLPKGKVLDVLRRYQEALRAQ GALDFGDILVYALRLLEENPEVLAKVRKRARFIHVDEYQDTSPVQYRFARLLAGEEANLMAVG DPDQGIYSFRAADIRNILDFTRDFPGARVYRLEENYRSTEAILRFANAVIQKNRLRLEKTLRPVKP GGEPVRVYAAPEAREEAREVAEEIFRLGPPYERFAVLYRTNAQSRLLEQALAAKGLPYRVVGGV GFFERAEVKDLLAYARLSLNPEDGVSLKRVLNTPPRGIGPATLARLEALAQAEGVPLLGAIRLGA ERFPKPEPLRAFLALLDELADLAFGPPEAFFRHLLSATDYLQYLKEHHPEDAEDRLENVEELLRA AKEAQDLQEFLDRVALTARADQDGGRGVALMTLHNAKGLEFPVVFLVGVEEGLLPHQSSLST LEGLEEERRLFYVGVTRAQDRLYLSYAREREVYGRREPRRMSRFLEEVPEGLYLPHDPYRQGA QPKPAPRAQGAFRGGEKVVHPREGPGTVVAASGDEVIVHFEGVGLKRLSLKYADLRPA K7QWX5 MASSLSKAELVPTPEQEKALHLYRSRQDFKLVAVAGSGKTTTLRLMAESFPRRHIAYLAFNRA 86 MKEEARRKEPPNTRVFTLHALAYRRTVPGTPYEAKERLGNGQVRPVHVRERLQVDPLLAYVV RSGLERFIRSGDPEPLPRHLPRDWRKTVEARGPSGFAEVERAVKGVALLWKAMRDPKDPFPL SHDGYVRIWREEGAGGDPPAGVILVDEAQDLDPNFLTVLSGWRGKAQQVFVGDPRQQIYG WRGAVNAMGEIDLPESPLTVVSFRFGEPLASFVQAVTARQTQGLVPLVGRAGWATEVHVNL EPTPPFTILTRSNLGLVTALLEGAQLFSLQKEEAHWGGVEELVWLLTDLQAIKEGGERPRPHPE LLGISKWEEVESLAEYSIVLNRLLRLAKEYDLEALAHKIAQLEIGPEEGAKLVLSTAHKAKGREWD RVLLWEDFYWVAAYRWFFPNTAPPPSEPSPEFLEEENIFYVAMTRARLGLHISLPEALAEEEAK RILDRLSQGVPSGEDRGEDERGETLPAPFTGPTPVSPKEATFPLPSLYDRLLSEALNGGRDPLLH LLRDDLARLSALSPTPLPPEVAQALWERARPEEALGAIREGLGAMWREDPYELLRAINALALLG GRNPRKLAKILGDRFPGGEEAEDLLFVARARKRELMGRSLAEFWRGLGASVRHPLLKAYARARS K7QTS9 MRLYVASAGTGKTETLMGELKALLEGGVPLRRVAAVSFTRKSAEELRLRVRRLLEAHREAFWA 87 REALREVHGALFTTLHGFMAEALRHTAPFLGLDPDFRVMDGFLAQALFLEEARSLLFLEGHPE APELLELLEALYEKRSLAEAFTPLPGAEGLLALYERVLARYRARTQEVLGPGDLEAKALLLLRHPE ALGRVAERFSHLLVDEFQDVNPLQGRFLRALEEAGVRWAVGDPKQSIYLFRNARVEVFLRAR AAAEEVRALSRTHRHAKQVVELLNRFTTRFFRAEEGNRVEGVREAEGRVEVHWVLGKLEEAR RAEARLLAQRLLALRAEGIPFGEMAVLVRARTSLPPLEKALRAAGVPFVRGRGQSFFARPEVR DLYHALRLALAERPYALEDRLSLLAFLRSPFLGLDLSELEEALRAEDPWPLLPKGVQEALEGLRAL ALLPPLEALRRLARDEGFLRRISRRARANLDTLLLLAAGAREPTLEDLLLWLALRAKDPESVELPE GGGGVTLLTVHGAKGLEWPWALYDVSRGPSERPPPLLVDEEGRVALKGTEAYRALLKEAERA EREEALRLLYVALSRARDLLLITGSTSQRPGPWAEALQALGLGPDAQDPWVETHPLEAIPPLPPI PQAPQDPRPAPYTPWRGEPRARPPWSPSAHLKAEAEPLEVLGEGEALPEWARAVGTLVHY AIARHLDPEDEGAMGGLLRQEVALAFGEGEREALLEEVRALLRAYRSLLSGALPPLEARAEDHA ELPLLLPHKGTVWYGVLDRLYRVGDRWYLDDYKTDQKVRPEAYRFQLALYRKAVLEAWGVE AEARLVYLRHRQWPLSPAELEAALEGL D1AF88 MSSSQVTGRPTIVKDAEIAVEQRRVDQAHARLEEMRAEAQAMIEEGYRQALAGTKGSLVDR 88 DAM\NQAALRVQALNVADDGLVEGRLDLADGQTRYIGRIGVRTRDHEPMVIDWRAPAAEA FYRATPEDPQGWRRRVLHTRGR-R/VDLEDDLLDPSAADSLTIVGDGAFIASLARTREGTMRD IVATIQREQDEVIRAPADGTVLVRGAPGTGKTAVALHRVAYLLFRHRRRFGSRGVLWGPNRR FTAYIERVLPSLGEGSATLRSLGDLVEGVSATVHDPPELAALKGSAAMAPVLRRAVTDHPPGA PDKLRWHGGVWELGRPQLDKLRTSLHRRSTGSVNASRRRVAEALLDALWERYVHTGGTEP EPDEPVQGELALWEGILAEGGLAPLDEQDRPSSPADRTSREAFVKNVREQRAFTDFLTAWW PIRRPLDVLRSLGDAARLRRAAGRDLDRAQVELLAASWRRALAGDPPTLSYQDIALLDEIDALL GPPPQPSRATAREEDPYVVDGIDILTGEWADEDWEPGLQELTTTERLERARRVDDEVADVR PEYAHIWDEAQDLSPMQWRMLGRRGRQATWTIVEDPAQSAWEDLEEARKAMEAALDGP AARRGRSRRPRRRPRHEYELITNYRNTTEIAAVSARVLRLALPEARPARAVRSSGHRPVIDLVP EEELQAAARRAVRTLLECIVEGTIGVIVPLPGDAWGESDRRALSAGFPERVQVLDVLEAKGLEF DAAVICAPETIAAQSPRGLRVLYVAVSRATQRLTVLTADPWVRRRLAGGESAR F8A884 MTSISLDQYQECtAVKAKGNTLWAGPGAGKTRVLLAKAIHLLEQGIDPEKVLILTFTIKTTQELK 89 ERLASIGIKGVKVDTFHALAYDLLKAKGIKPRLATEEELKALARDLSKRKGLSLKDFRKALDKGEN HYRSLWEEALKLHGLYDFSLLLKEATGHYLQQEKVYLLIDEFQDLNPELTSFLKTFTKAEFFLVGD PAQAIYGFRGACPQVIKEFVDYLAPQIYFLKKSYRVPEKVLNFAETLRETQGFPLEPLEAVQKGG NRLGLSFNKPFNEAKGVAKLVSELLGGLQMEASQRGLAPPEIAILSRVRTLLNPIKEAFIKFGIPF QVPSENLKEEISAIESLSDIAKSIKSLKELEAYLAEGPSSVKEAWLESQSLEGFLFRLEMLKTFASISI RKDGVPLLTIHEAKGLEFKWILVGAEDGLPFTLLEDYDLAEEKRVAYVAVTRAQESFYFTQVK TGRFLYGHKLSGKVSPFFETLPIKEKSSKTKPKARQKKLFG A0A087LEB0 MTISVIDELLEKNKQNMNKTAKDAVEAQUAYAKKEVKKLQEIRPHPYFGRLDFEDEFGRETIYI 90 GKKGLEKDGELIWDWRTDLGRLYNAYQGVQKTFQIGKENRPVTIHGKRGIVIKNGKVIKVTDI GKSEIIENDNGEKVKYMDDYLKEILTNTEEAHRLRDIIASIQAEQDEIIRLPLKDTIIVQGAAGSGK STIALHRISYLLYQYHEQVKPKDILILAPNElFLSYIKDIVPEIElEGIEQRTFYDWASTYFTDVHDIPD LHEQYVHIYGSTEKEDLIKIAKYKGSLRFKKLLDDEVEYIGNTMIPHGDWIESGVILSKEEIHQFY HAKEHLPLNVRMKEVKEFIINWRNEQINIRKQQIEDEFEEAYRKWVVTLPEGEERKAVYEALEK AKQLRMKIFQEKMQHEISLIVKKMENIPALLMYKSVFQKKVFEKFHPDIDEELLSLLLKNGRQIK QERFMYEDIAPLIYLDAKINGKKLQYEHIVIDEAQDYSPFQLAIMKDYAKSMTILGDIAQGIFSFY GLDRWEEIESYVEKEKEFKRLHLQTSYRSTKQIMDLANRVLLNSNYDEPLVIPVNRPGDVPTIKK VESIGELYDEIVNSIRIFEEKGYKKIAILTASKQGAIDTYDQLMRRQITQMEVITEGHQALKEKIVII PSYLVKGLEFDAVIIEDVSDETEKDETQHAKMLYMSITRAHHDLHLFYRGNISPLLEERDPSAPP KPRKSFADWLITDINDPYVEPQVEAVKRVKKEDMIRLFDDEEEEFVEEAFEDDRERYYDFHAW LKVWRRWAEMRKQLDEKS B5Y6N2 MALPQENLIPPSPSHNHLTLSLRSHIGGFFIYNEDVDSVDLSKLNEAQKQAVTAPPKPLAIIAGP 91 GSGKTRVLTYRALFAVKEWHLPPERILAITFTNKAADELKERLGRLIPEGDRIFAATMHSFAAR MLRYFAPYAGISQNFVIYDDDDSKGLIEDILKQMNMDTKRFRPNDVLNHISAAKARMFDCNT FPEFIRQRYGSWGYYFDTVHQVFMTYERLKEQSQALDFDDLIMVLAQRMEDRPELREMIAGL FDLVMVDEFQDTNFAQYQMLLYMTNPHYSGMNNVTIVGDPDQSIYGFRAAEYYNIKRFIDD YNPEVVFLDLNYRSNRTIVDSASALINDSPSALFERKLESIKGAGNKLILRRPFDDADAAITAAFE VQRLFIKMGIPYEEIAVLMRTRALTARVEREFATRNIQYHIIGGVPFFARREIKDILAYLRLSRNA MDRVSLKRILTMKKRGFGTASLEKLFNFAEENKLTLLEAMKAAVESLLFKKLSMNDYLESLYTLI QTIQEIAEPSQAIYLVMEQENLLDHFRSISKSEEEYIERTENVKQLISIAEESADMDDFLQRSALG TRENNGGVEGVAISTVHGVKGLEFQAVILYYVTDGFFPHSLSVTTAEKEEERRLLYVAMTRAKE HLIFYVPYKQPWGNGFEQMARPSPFLRSIPKELWDGKPNEIESLYAPYSPQQKWSE D7BJL0 MNDPIRHKEGPALVFAGAGAGKTRTLTQRVKWLVEEGEDPYSITLVTFTNKAAGEMKERIAR 92 LVEAPLAEAVVNGTFHRFCLQSLQVYGREIGLEKVAVLDSAAQRKLAERIIAGLFPAKPPRGFT PMAALGAVSRAANSGWDDIQLATMYADLTEKIVNFRWAYEEAKKGLGALDYDDLLLRGVRL LKLSEGAARMVRRRAAYLMVDEFQDTNGVQLELVRAIAPGTSPNLMVIGDPDRSIYGWRG ANYRTILEFRQHYPGAAVYGLYTNYRSQAGWEVANRIIAQNATRKPEMQEAHLPQSEEPFLL VAKNRWEEAHEVAQAVEFYRGQGIALEEMAVLMRANFLSRDLEQALRLRGIPYQFTGGRSFF ERREIQLGMAVLKVLANPKDSLAVAALVEEMVEGAGPLGIQKVLEAAKAANLSPLEAFRNPA MVKGLRGKEVQAEAMRLAEVLQDQVARLAAEAPEYHALLKETLDRLGFEAWLDRLGEESEQ  VYSRKANLDRLLQGMQEWQEVNPGAPLQDLVGTLLLEAGDTPAEEGQGVHLMTVHASKG MEFRVVFVIGLNEGLFPLSKASSSFEGLEEERRLMYVAVTRAKEVLHLSYAADGVVSRFAQEAR VPVEEYDPRLGWSGRQNQQALKALLEIA E8MZN5 MDSLEHLNPQQRAAVTASAGPVLVLAGPGSGKTRVLTFRIGYLLSQLGVAPHHILAVTFTNKA 93 AREMQSRVEKLLGHSLQGMWLGTFHAICARILRREQQYLPLDANEVIEDEDDQQALIKRALR DLNLDEKLYRPTSVHAAISNAKNNLILPEDYPTATYRDEVVARVYKRYQELLVSSNAVDFDDLLL YAWKLLNEFSTVREQYARRFEHILVDEFQDTNLAQYELVKLLASYHRNLEVVGDEDQS1YRWR GADYRNVLRFEEDEPDRQKILLEQNYRSTQRVLDAAQAVINRNRNRTPKRLKSTPEHGEGEKL VLYEAVDDYGEAAFVVDTIQQLVAGGKARPGDFAIMYRTNAQSRLLEEAFLRAGVPYRLVGA MRFYGRREVKDMIAYLRLVQNPADEASLGRVINVPPRGIGDKSQLALQMEAQRTGRSAGLIL MELGREGKDSPHWQALGRNASLLADEGSLLGEWHRLKDEISLPSLFQRILNDLAYREYIDDNT EEGQSRWENVQELLRIAYEYEEKGLTAFLENLALVSDQDTLPENVEAPTLLTLHAAKGLEFPIVFI TGLDDGLIPHNRSLDDPEAMAEERRLFYVGLTRAKKRVYLVRAAQRSTYGSFQDSIPSRFLKDI PADLIQQDGRGRRMGRSWQSESRRSWDDNYAGDVIGSRPERAKPSHAPILQPREKPGMRV KHPSWGEGLWDSRIQDEDETVDIFFDSVGFKRVIASIANLEILS L0INW7 MDINGQIIKLNRNKTQGTLKLTNGQKIKFKINSDSVKPIFLYEYYKFKGNMIEDTLIIDDIYGIAND 94 ININDFTELEPSVAHDKINNICNRENVLHVGNLIDLINDENFITVVNDTIGEEKATIFLSNLQKIKD RQEYIDVWDIIKKTNPTEDINVPIKIVNALKYRASMNNITVSQLIKESPWIIEQLDIFDSITERKKIA ENIATHYGLSNDSNKAVISYAIAMTNNYIQQGHSYIPYYTLVSRVSNSLKLDENKVNDTLKFLPN DNKSGYLIRDNKYKDEIENEYNSDKKIGYSVYLPKIEHMEKYIADIISSILKKKSTINKIELQKNLKLY RSENKLIFSKEQEEAIFSISDNKITVITGGAGTGKITVIKAIIDLVNKMGYTPWLAPTGIASQRVA PNVGSTIHKYARIFDTYDPVFDEIEENKENNSGKVIIVDEMSMITVPVFAKLLSVTLDADSFIFVG DPNQLPPIGAGGVFEALIELGNKNINNINTWLNQSFRSKNSIVKNAQN1LEDKPIYEDDNLNIIE AKSWNKIADEWNLIRKLLDNGVQYSDIMVLSSKRGEGKNGVSLLNERIRKEIENNKGKYAVG DIVITTRNDYDNKSSYFRSKELKKYINSIRHEERPTIENGTVGVIKDISDNEVIIEYNTPMPVEAKY NMEELDVVYIEYGFAITVHKAQGGQAKYIIFASDEPRNISREMLYTAITRCKNGKVFLIGGENED WKIKKEHSFVLSKLKYRILDNIHQQEKESKINSKIVLINQ D3PR99 MSDLLSSLNPSQQEAVLHFEGPALWAGAGSGKTRIVVHRIAYLLRERRVYPAEILAVITTNKA 95 AGEMKERLEKMVGRPARDLWVSTFHAAAVRILRTYGEYVGLRPGFVIYDEDDQNTLLKEVLK ELELEAKPGPFRAMIDRIKNRGAGLAEYMREAPDFIGGVPKDAAAEVYRKYQSGLRMQGALD ENDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVQYKLTRLLAGERPNLMWGDPDQ SIYGERSADINNILDFTKDYPGARVIRLEENYRSSSSILRVANAVIEKNALRLEKVLRPTRPGGEPV RLYRAPNAREEAAFVAREIVKLGNFQQIAVLYRTNAQSRLLEEHLRRANVPVRLVGAVGFFER REIKDLLAYGRVAVNPADSINLRRIVNTPPRGIGATTVSRLVEHAQKTGTTVFEAFRVAEQVISR PQQVQAFVRLLDEL1EAAFESGPTAFFQRVLEQTGFREALKQEPDGEDRLQNVEELLRAAQD WEEEEGGSLSDFLDSVALTAKAEEPQGDAPAEAVTLMTLHNAKGLEFFIVELVGLEENLLPHR NSLHRLEDLEEERRLFYVGITRAQERLYLSYAEERFYGKREYTRPSRFLEDIPQDLLKEVGAFGD SEVRVLPQARPEPKPRTQLAEFKGGEKVRHPKEGSGTVVAAMGGEVIVMFPGVGLKRLAVK FAGLERLE D3PLL2 MKVRVASAGTGKTASLVLRYLELIAKGTPLRRIAGVTFTRKAADELRVRVAAAIEEVLQTGRHLS 96 EVASGGSRAAFQEAAREIAGATLSTIHGEMAQCLRLAAPLHLDPDFSMLGDWEAQAIFEEE WQTLRYLAQDAHHPLFGLVSDELTEPLLHLFSRRSQAEVFEPAAGEANQHLLQVYQTVYAAY EARLGANLLSPSELERKALELARNDRAMKRVLERVRVLLVDEYQDVNPVQGAFFAALEQARLP IEIVGDPKQSIYAFRNADVSVERKALREGKSEPPLTHSYRHSRVLVRELNGLIGYLAKEGLGEGLE EAPPVEGVRPEQGRLEVHWVVGELPLEELRKQEARVLAGRLAALRGPIEYSQMAVLVRSYGS VRFLEEALAEAQIPYVLLQGRGYYERQEVRDLYHALRAALDPRGLSLAVFLRSPFGQHTEAGPL KPLELPQIEGVLRADDPLGRLAQHWPSVYERLRQIQAQVRLMAPLEVLKFLIRAPLMDGRPYH DFLEPRARENVDALLFYFAPRPPQNLEGLERLELLSRQADAGDVPQSGEGVQILTVHQAKGL EWPLVAVFDLGRMNVHRPQPLYLGQGPNGGDGGRLRRVVVALPETPQFEAFRQQVKLQEE EESYRLYVAASRARDTLLTASASHGQPEGWGKVLEAMNLGPASKPYHRPDFHLQTWPYQ PAPPVRVLSQPAPLQPSPWVDARFEPEPFPPLESPSALKRLEAEPLPLPDPEEGEAVPGRARAI GTLVHYAIGQNWRPDNPQHLANLEAQEVMFPFGPDERRGIMAEVQALLEHYQELLGRALP WPRDEDYPEFAVALPLGSTVWQGVIDRLYRVGQQWYLEDYKTDQEMRPERYLVQLGIYLAA IRQAWQIEPEVRLVYLRFGWVERLDKAILEAALGEIMPKGEGLRR Q9RTI9 MTSSAGPDLLQALNPTQAQAADHFIGPALVIAGAGSGKTRTLIYRIAHLIGHYGVHPGElLAVT 97 FINKAAAEMRERAGHLVPGAGDLWMSTFHSAGVRILRTYGEHIGLRRGFVIYDDDDQLDI1K EVMGSIPGIGAETQPRVIRGIIDRAKSNLWTPDDLDRSREPFISGLPRDAAAEAYRRYEVRKKG QNAIDEGDLITETVRLFKEVPGVLDKVQNKAKFIHVDEYQDTNRAQYELTRLASRDRNLLWG DPDQSIYKFRGADIQNILDFQKDYPDAKVYMLEHNYRSSARVLEAANKLIENNTERLDKTLKPV KEAGQPVTFHRATDHRAEGDYVADWLTRLHGEGRAWSEMAILYRTNAQSRVIEESLRRVQI PARIVGGVGFYDRREIRDILAYARLALNPADDVALRRIIGRPRRGIGDTALQKLMEWARTHHTS VLTACANAAEQNILDRGAHKATEFAGLMEAMSEAADNYEPAAFLRFVMETSGYLDLLRQEG QEGQVRLENLEELVSAAEEWSQDEANVGGSIADFLDDAALLSSVDDMRTKAENKGAPEDAV TLMTLHNAKGLEFPWFIVGVEQGLLPSKGAIAEGPSGIEEERRLFYVGITRAMERLLMTAAQN RMQFGKTNAAEDSAFLEDIEGLFDTVDPYGQPIEYRAKTWKQYRPTVPAATTAVKNTSPLTAE LAYRGGEQVKHPKFGEGQVLAVAGVGERQEVTVHFASAGTKKLMVKFANLTKL M1E5C5 MDLNLNEDQKRAVYSDSRALLIVAGAGTGKTRVLITRAARLIKENPDARYLLLTFTKKAAREM 98 TTRVRELIEEDTKNRLYSGTEHSFCSNIIRRRSERVGLTNDEVIIDESDSLDLMKKVESRIYSKEKID  SLIFKPKDILSLYSYARNNNQDFIEIVQRKYKYVNFEDIKKIISLYELNKKERNYLDFDDLLMYGLLA IKTLEKSPFDEVLVDEFQDTNQIQAEMLYYFYDLGSRISAVGDDAQSIYSFRGAYYENMENFIKR LDAEKIILSSNYRSTQQILDIANSIIQSSYSSIKKELVANVRLKENVKPKLVIVSDDWEEARYVARE MQKFGEKGLKVAALYRAAYIGRNLESQLNSMGIKYSFYGGQKLTESAHAKDFIVISFLRVEVNP KDEIALIRILKMFPGIGEKKAEKIKDAVISGDNLKKALSKEKNLEELNIFFDKLFKITDWHDLLELVF DFYKDIMNRLYPENYEEREEDLIKFMDMSSNYDNLVEYLEAFTLDPVEKSEEDNNNVILSTIHS AKGLEFDWELLSVIESVYPHFRAQSTDEIEEERRLFWAITRAKQRLIFTFPRHSKKSRGYFAKNTI SPFLREKDNYLEVFIAR Q5SIE7 MSDALLAPLNEAQRQAVLHFEGPALWAGAGSGKTRTVVHRVAYLVARRGVEPSEILAVTFT 99 NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEV LKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALD FGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLIAGEEANLMAVGDPDQG lYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPV RLYRAEDAREEAREVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARWGGVGFFE RAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAART FSRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPAYLREAYPEDAEDRLENVEELLRAAK EAEDLQDFLDRVALTAKAEEPAEAEGRVALMTLHNAKGLEFPWELVGVEEGLLPHRNSVSTL EGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPP HRPRPGAFRGGERWHPREGPERIVAAQGDEVIVHFEGFGLKRLSLKYAELKPA B5YD55 MNNQFDSEKKIFIIPSRKKKEFLERIEKDLNEEQRKWLEADGPSLVIAGPGSGKTRTIVYRVGYL 100 VALGYSPKNIMLLTFINQAARHMINRMALIRESIEEIWGGTEHHVGNRILRVYGKIIGINEQY NILDREDSLDLIDECLEELFPEENLGKGILGELFSYKVNTGKNWDEVLKIKAPQIIDKIEIVQKVFER YEKRKRELNVLDYDDLLFFWYRLLLESEKTRKILNDRFLYILVDEYQDTNWLQGEIIRLTREENKN ILWGDDAQSIYSFRGATIENILSEPEIFPGTRIFYLVENYRSTPEIINLANEIIKRNTRQYFKEIKPVL KSGSKPKLVVVVRDDEEEAQFWEVIKELHKEGVKYKDIGVLERSNYHSMAVQMELTLQGIPYE VRGGLRFFECtAHIKDMISLLKILFNPQDEISAQRFFKLFPGIGRAYAKKLSQVLKESKDFDKIFQ MQFSGRTLEGLRILKNIWDKIKVIPVQNFSEILRVFFNEYYKDYLERNYPDFKDREKDVDQLILLS ERYDDLEKELSELTLYTYAGEKLLEEEEEEKDEWLSTIHQAKGLEWHAVFILRLVQGDFPSYKS MDNIEEERRLFYVAVTRAKRELYVITYLTRKVKDMNVFTKPSIFLEELPYKELFEEWIVQBEI MLSPEGGEEETKAIPLEEEILLAWRVESAALPPNFLAPVSASLHTLVREAEGKEGAELEAYAWER F6DJA4 LEELARTSWKDAIQSFLEVAAEKPEVLRAGLLWERTWNRLSPEEREALYRKAERFKPTAELASK 101 ASFLQGPPPPPKPLSPSVQAARSSPPRFTPTPEQEEAVRAFLSREDMKLVAVAGSGKIIILRL MAQSAPKERLLYVAFNRSVRDEAERTFPGNVEVLTLHGLAHRHWRGSGAYQRKLAARNGR VTPGDVLEALELPRERYALAYVIRSTLEAFLRSASEVPTPAHIPPEYREVLQRRDKDPFSERYVLK AVRLIWKLMQDPDDSFPLSEDGFVKIWAQAGAKIRGYDAVLVDEAQDLSPVFLQVLEAHRGE LRRVYVGDPRQQIYGWRGAVNAMDKLDAPERKLTWSFREGEDIARGVRRFLAHVGSPIELH GKAPWDTEVSLARPEPPYTALCRTNAGAVEAVISELLEEGREGARVFWGGVDEIAWLLRDA HLLKVGGEREKPHPELALVENWEELEELAKEVNHPQARMLVRLARRYDLLELARLLKHAQADE EGKADLWSTLHKAKGREWDRWLWGDFIPVWDEKVREFYRKQGALDELKEEENVVYVALT RARRFLGLDQLPDLHERFFQGEGLVKPPSVSPLSVGGAGVSADLLRELEVRVLAKLEDRLKEVA EVLAALLVEEASKAVAEAMREMGLLGEEG F6DIL2 MSDALLAPLNEAQRQAVLHFEGPALWAGAGSGKTRTWHRVAYLVARRGVFPSEILAVTFT 102 NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFWYDEDDQTALLKEV LKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALD FGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQG IYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPV RLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARWGGVGFFE RAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAART FPRAEPLRHFVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRLENVEELLRAAK EAEDLQDFLDRVALTAKAEEPAEAEGKVALMTLHNAKGLEFPWFLVGVEEGLLPHRNSLSTLE GLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPH RPRPGAFRGGERWHPRFGPGTVVAAQGDEVTVHFEGVGLKRLSLKYAELKPA F6DJ67 MEANLYVAGAGTGKTYTLAERYLGFLEEGLSPLQWAVTFTERAALELRHRVRQMVGERSLG 103 HKERVLAELEAAPIGTLHARVCREFPEEAGVPADFQVMEDLEAALLLEAWLEEALLEALQ DPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRALRRRMEELFHG ERPEERYPGFPKGWRTEEPEWPDLLAWAGEVKFNKKPWLEYKGDPALERLLKLLGGVKEGF SPGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRALEREEVRAYYRGRER RLLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQS1YSFRRADPKVMERFQAEAAKEGL RVRRLEKSHRYHQGLADFFINRFFPPLLPGYGAVSAERKPEGEGPWVFEIFQGDLEAQARFIAQ EVGRLLSEGFQVYDLGEKAYRPMSLRDVAVLGRIWRDLARVAEALRRLEVPAVEAGGGNLLE TRAFKDAYLALRFLGDPKDEEALVGLLRSPFFALTDGEVRRLAEARGEGETLWEVLEREGDLSA EAERARETLRGLLRRKALEAPSRLLQRLDGATGYTGVAARLPQGRRRVKDWEGTLDLVRKLEV GSEDPFLVARHLRLLLRSGLSVERPPLEAGEAVFLLTVHGAKGLEWPWFVLNVGGWNRLGS WKNNKTKPLFRPGLALVPPVLDEEGNPSALFHLAKRRVEEEEKQEENRLLYVAATRASERLYLL LSPDLSPDKGDLDPQTLIGAGSLEKGLEATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPIA ARDPEAARRRLLGEPEPEGGEAWEPDGPQETEEEVPGGAGVGRMTHALLERFEAPEDLERE GRAFLEESFPGAEGEEVEEALRLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVG EDWVLDYKTDRGVDAKAYLLQVGVYAIALGKPRALVADLREGKLYEGASQQVEEKAEEVLRR LMGGDRPEA G8N9P8 MDAFPSGKPLDEAWLSSLNEAQRCtAVLHFEGPALWAGAGSGKTRIVVHRVAYLMARRGV 104 YPSEILAVITTNKAAEEMRERLKAMVKGAGELWVSTFHAAALRILRFYGERVGLKPGFVVYDE DDQTALLKEVLKELGVSAKPGPIKALLDRAKNRGEPPERLLADLPEYYAGLSRGRLLDVLHRYQ QALWAQGALDFGDILLLALKLLEEDPEVRKRVRKRARFIHVDEYQDTSPVQYRLTKLLAGEEAN LMAVGDPDQGIYSFRAADIKNILQFTEDFPGAKVYRLEENYRSTERILRFANAVIVKNALRLEKT LRPVKSGGEPVRLFRARDAREEARFVAEEVLRLGPPYDRVAVLYRTNAQSRLLEQALASRGIGA RWGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATVEKVQA1AQEKGLPLY EALKVAAQVLPRPEPLRHFLALMEELMDLAFGPAEAFFRHLLEATDYPAYLKEAYPEDLEDRLE NVEELLRAAREAEGLMDFLDKVALTARAEEPGEAGGKVALMTLHNAKGLEFPWFLVGVEEG LLPHRSSVSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRPEASRPSRFLEEVEEGLYEEY DPYRLPPPKPVPPPHRAKPGAFRGGEKWHPREGLERNAASGDEVIVHFDGVGLKRLSLKY ADLRPA Q1J014 MPDLPASSLIAQLNPNQAQAANHYTGPALVIAGAGSGKTRTLVYRIAHLIGHYGVDPGEILAV 105 TFTNKAAAEMRERARHLVEGADRLWMSTFHSAGVRILRAYGEHIGLKRGFVIYDDDDQLDIL KEIMGSIPGIGAETHPRVLRGILDRAKSNLLTPADLARHPEPFISGLPREVAAEAYRRYEARKKG QNAIDEGDLITETVRLFQEVPAVLERVQDRARFIHVDEYQDTNKAQYELTRLLASRDRNLLWG DPDQSIYRFRGADIQNILDFQKDYLDAKVYMLEQNYRSSARVLTIANKLIENNAERLEKTLRPVK EDGHPVLFHRATDQRAEGDFVAEWLTRLHAEGMRFSDMAVLYRTNAQSRVIEESLRRVQIP AKIVGGVGFYDRREIKDVLAYARLAINPDDDVALRRIIGRPKRGIGDTALERLMEWARVNGTSI LTACAHAQELNILERGAQKAVEFAGLMHAMSEAADNDEPGPFLRYVIETSGYLDLLRQEGQE GQVRLENLEELVSPAEEWSRENEGTIGDFLDDAALLSSVDDMRTKQENKDVPEDAVTLMTL HNAKGLEFPWFIVGTEEGLLPSKNALLEPGGIEEERRLFYVGITRAMERLFLTAAQNRMQYGK TLATEDSRFLEEIKGGFDIVDAYGQVIDDRPKSWKEYRPTESARPGAVKNTSPLTEGMAYRGG EKVRHPKFGEGQVLAVAGLGDRQEVTVHFPSAGTKKLLVKFANLTRA Q745W4 MALRPTEEQLKAVEAYRSGQDLKWAVAGSGKITTLRLMAEATPGKRGLYLAFNRSVQQEA 106 ARKFPRNVRPYTLHALAFRMAVARDEGYRAKFQAGKGHLPAQAVAEALGLRNPLLLHAVLGT LEAFLRSEAASPDPGMIPLAYRTLRAGTKTWPEEEAFVLRGVEALWRRMTDPKDPFPLPHGA YVKLWALSEPDLSFAEALLVDEAQDLDPIFLKVLEAHRGRVQRVYVGDPRQQIYGWRGAINA MDRLEAPEARLTWSFRFAETLARFVRNLTALQDRPVEVRGKAPWATRVDAALPRPPFTVLCR TNAGWGAWVTHEVHRGRVHWGGVEELVHLLRDAALLKKGEKRTDPHPDIAMVETWEE LEALAEAGYAPAYGVLRLAQEHPDLEALAAYLERAWTPVEVAAGWVSTAHKAKGREWDRV VLWDDFYPWWEEGWRVNWGSDPAHLEEENLLYVAATRARKHLSLAQIRDLLEAVDRMG VYRVAEEATRAYLLLSAEVLRGVATDPRVPAEHRVRALKALGYLERGEEALDSPGKPGGQG Q721S0 MSDALLAPLNEAQRQAVLHFEGPALWAGAGSGKTRTVVHRVAYLVARRGVEPSEILAVTFT 107 NKAAEEMRERLRGLVPGAGEVVVVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEV LKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALD FGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQG IYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPV RLYRAEDAREEAREVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARWGGVGFFE RAEVKDLLAYARIALNPLDAVSLKRVLNTPPRGIGPATWARVQLIAQEKGLPPWEALKEAART FPRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPAYLREAYPEDAEDRLENVEELLRAAK EAEDLQDFLDRVALTAKAEEPAEAEGRVALMTLHNAKGLEFPWFLVGVEEGLLPHRNSVSTL EGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPP HRPRPGAFRGGERVVHPREGPGTVVAAQGDEVEVHFEGFGLKRLSLKYAELKPA F2NK78 MDLLRDLNPAQREAVQHYTGPALVVAGAGSGKTRIVVHRIAYLIRHRGVYPTEILAVTFTNKA 108 AGEMKERLARMVGPAARELWVSTFHSAALRILRVYGEYIGLKPGFVVYDEDDQLALLKEVLG GLGLETRPQYARGVIDRIKNRMWSVDAFLREAEDWVGGLPKEQMAAVYQAYEARMRALG AVDFNDLLLKVIGLFEAHPEVLHRVQQRARFIHVDEYQDTNPAQYRLTRLLAGAERNLMVVG DPDQSIYGFRNADIHNILNFEKDYPDARVYRLEENYRSTEAILRVANAVIEKNALRLEKTLRPVRS GGDPVFLYRAPDHREEAAFVAREVQRLKGRGRRLDEIAVLYRTNAQSRVLEEAFRRQNLGVRI VGGVGFYERREVKDVLAYARAAVNPADDLAVKRVLNVPARGIGQTSLAKLSQLAETARVSFFE ALRRAGEVLARPQAQAVQREVALIEGLANAAYDTGPDAFLRLVLAETGYADMLRREPDGEAR LENLEELLRAAREWEEQHAGTIADFLDEVALTARAEEPEGEVPAEAVTLMTLHNAKGLEFPVV FIVGVEEGLLPHRSSTARVEDLEEERRLFYVGITRAQERLYLTLSEERETYGRREAVRASRFLEDIP EAFLQPLSPFGEPLGAGREPVAVRPTRRSSAAGGFRGGEKVRHPRFGQGLVVAASGEGDRQE VTVHFAGVGLKKLLVKYAGLERIE

TABLE 18  >tr|L0B9N8|L0B9N8_9EURY UvrD Rep helicase SFIOS = Thermococcus sp. EXT9 GN = e9a-1 PE = 4 SV = 1 (SEQ ID NO: 58) MSEALPVISFEFSLPEESVIKIYGPPGTGKTTILVRIIEHLIGETHDHTEFLESYGLSLLF GQYGAEDVIFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAIDGV ITQNFGSLSPEDINFRLFQRQNGLRFESSEMGYSNVENDGNRLWNALTWAYNVYYPTKGPK ARHEALKRLAFKLWKYPPLWEEYKTEKGILDYNDMINKAYEGLKSGEIDPRNLPGHKYSP KVLIVDEFQDLSPLUEIERLLANYMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVL KRSYRLPIVVQAKAMTVISKTRHRKEKTVAPRIDLGDFKYKLFWEPDFLNDLVREAUGH SIFILVRTNRQVLKLGKELILAGVHFRHLKVDYRSIWEAGSKEWGTERDLVQALLKARRG EELEIADLVTILYYSELIDWHLGEKLPEKERYKKIAEQMEKTIEAIEKGLMPFDILKVKD DPFSVLDLEKIESLSPRHGKVAVELIREIMKEKSQVEVPRDAEITLDTLHASKGREADV VFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKKVYLLNGKEPFPVL >tr|L0B90|L0B9J0_9EURY UvrD Rep helicase SFIOS = Thermococcus sp. IR148 GN = 148-1 PE = 4 SV = 1 (SEQ ID NO: 59) MRVKIYGPPGIGKITTLQRTIDYTLGNSSEPPIPLPESEPTDLEPKNLAFVSFINTAIDV IGKRTGITTRSKEAPYMRTIHGLILSVLAEHFDPVAVDNLGKLADIQAEFSMRMGYYYSK DPFEFAEGNMKENVITRALELYLPKTGDVEEALKLIDNREDRKFALAWYRYKRQKKIMDF DDILVIGYEHLEDFYVPVEVAFIDEGQDNGPLDYILLEKGFEGAKFVFLAGDPLQSIYGF KGADPRLEVRWKADKETILPRBYRLPKKVWLLSQSWAISLGIKGAVVRYAPSEKLGRVSR MKFIEALSYAVEQAKRGRSVLILARTNSLVKFVGNILSIEFGVAYGHLKRASYWESHLLK FIEGLQMLKLWDGVTPIKVQDTKPITGLIRKLKDKHAREVLRRWRDSROISLEVQAVLQR IKKNPSEYFYITDFDRQALKAYFSKARLDLTEELIIDTIHAAKGEEADVVIFLDFIPTRS EERINPEELQEKLVAYVGFTRAREELIIVPIPAIKYEPMRDFMGVRQILGVVNEHKHLLI KELVGGL >tr|L0BAD9|L0BAD9_9EURY UvrD Rep helicase SFIOS = Thermococcus sp. IRI33 GN = i33-1 PE = 4 SV = 1 (SEQ ID NO: 60) MSEALPVTSFEFSLPRERIIKLYGAPGTGKTTILVKIIEHLIGFUHTEFLENYGINLPF GWEPGEVIENTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAVDGL ITQNFGSLSPEDINFRNFCRQNGLRFESSEMGYSNVENEGNQLWNALTWAYNVYYTTKGPK ARYEALKRLAFKLWKEPPLWEEYKKGRGILDYNDMTVRAYEGLRSGEIDPRNLPGHKYSP KVLIVDEFQDLSPLUEIFRLLANHMDLVTIAGDDDQTIFSYQGADPRLMNYVPGLEVVL RKSHRLPIVVQAKALTVISKTRHRKEKTVAPRIDLGDFKYKLFWFPDFLNDLVREAUGH SIFILVRTNRQVLKLGKELILAGVHFEHLKVDYRSIWEAGSKEWGTERDLVQALLKAKRG EELEVADLVTILYYSELIDWHLGEGISEKERYKKIAEQMEKTIEAIEKGLMPFDVLRVKE NPFSVLDLEKIESLSPRHGKVAVELIKELMKEKSQVVEIPKDARIYLDTLHASKGREADV VFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKKVYLLNGKEPFPVL >tr|L0BAT5|L0BAT5_9EURY UvrD Rep helicase OS = Thermococcus sp. AMT7 GN = a7-1 PE = 4 SV = 1 (SEQ ID NO: 61) MSEALSITSFDFTLPRERIIKIYGPPGTGKTTILVRIIEHLIGFUHTEFLENYGLSLPF GQYGAEDVIFMTRNSALKEFEARTGIKVKDRQNKPGRYYSTVEGIAFRLLIDSGAVDGL ITQNFQSLSPEDWFRHFCRQNGLRFESSEMGYSNIFNEGNQLWNALTWAYNVYYTTKGPK ARYEALKRLAFKLWKETPLWEEYKKEKGILDYNDMLTRAYEGLKSGEIDPRNLPGHKYSP KVLIVDEFQDLSPLUEIFRLLANHMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVL SKSYRLPIVVQAKALTVISKTRHRKEKTVAPRIDLGDFKYKLFWFPDFLNDLVREAUGH SIFILVRTNRQVLKLGKELILAGVHFEHLKVIDYRSIWEAGSKEWGTFRDLVQALLKAKKG EELEVADLVTILYYSELIDWHLGERISEKERYKKIAEQMEKTIEAIEKGLMPFDILKVKE NPFSVLDLEKIESLSPRHGKVAVELIKELMKEKSQNEIPKDAKITLDILHASKGREADV VFLINDLPRKWSNILKTREELDAERRVWYVGLTRARKKVYLLNGKHPFPIL >tr|W8NUG2|W8NUG2_9EURY Superfamily IDNA and RNA helicase and helicase subunits OS = Thermococcus nautili GN = BDO1 1302 PE = 4 SV = 1 (SEQ ID NO: 62) MNENEKLSKFIAKLKVL1EMERKAEIEAMRAEMRRLSGREREKVGRAVLGLNGKV-IGEEL GYFLVRYGREREIKTEISVGDLVVISKRDPLKSDLVGTVVEKGKRFITVALETVPEWALK SVRIDLYANDITFKRWLENLENLRESGRRALELYLGLREPEGGEEVEFTPFDKSLNASQR RAIAKALGSPDFFLIHGPFGTGKTR7LVELIRQEVARGNRVLATAESNVAVDNLVERIVD SGLKVVRVGHPSRVSRGLHETTLAYLMTQHELYGELRELRVIGENLKEKRDIFTKPAPKY RRGLTDRQILRLAEKGIGTRGVPARLIREMAQWLKINEQVQKTFDDARKLEERIAREIIR EADVVLTTNSSAGLEVVDYGSYDVAIIDEATQATIPSVLIPINRAGRFVLAGDHKQLPPT ILSEKAKELSKTLFEGLIERYPGKSEMLIVQYRMNERLMEFPSREFYDGRIEADESIRRI TLADLGVKSPEDGDAWAEVLKPENVIVFIDTARREDRFERQRYGSESRENPLEARLVKEA VEGILRLGVKAEWIGVITPYDDQRDLISSLLPEEIEVKTVDGYQGREKEVIVLSEVRSNR KGELGELKDLRRLNVSLTRAKRKLILIGDSSILSSHPTYRRIVEFVRERETVVDAKRIIG KVKIK >tr|B6YXQ7|B6YXQ7_THEONUvrD/REPhelicase OS = Thermococcus onnurineus (strain NA1) GN = TON_1380 PE = 4 SV = 1 (SEQ ID NO: 63) MIAPIPTTYSILGVAGAGKITQLIDLLNYLNFENSRNEKIWERHFEPVELNRIAFISFSN TAIQEIANRIGIEIKARKKSAPGRYFR7VTGLAEVLLYENNLMTFEEVRSVSKLEGFRIK WAREHGMYYKPRDNDISYSGNEFFAEYSRLVNTYYHVKSLSEIIEMHSKSHLLLDYIREK EKLGIVDYEDILMRAYDYRNDIVVDLEYMIIDEAUNSLLDYAILLPIAKNNATELVLAG DDAQLIYDERGANYKLEHKLIERSEIILNLTETRREGSEIANLATAIIDDMNYIQKREVL SAATHSTKVAHIDLFQ1vEvISILQMATTDLTVYILARTNAVLNYVAKVLDEYKIQYKKNER ITDFDRELLSLNRLMRNEYTNDDIYITYNYLRNKVAREEELKERLFQHKLHWTEKDVLGI LLLAYEQTTAKRILTTAKNINFKIKLSTIHSAKGSEADVVFLINSVPHKTKMKILENYEG EKRVLYVAVIRARKFLFIVDQPVARRYEQLYYIRSYESRAQGSLVNRVAVPVA. >tr|Q5JFK3|Q5JFK3_THEKO DNA helicase, UvrD/REPfamily OS = Thermococcus kodakarensis (strain ATCC BAA-918/JON12380/  KOD1) GN = 1K0178 PE = 4 SV = 1 (SEQ ID NO: 64) MNEKEVILSKFIAHLKELVEMERRAEIEAMRLEMRRLSGREREKVGRAVIGLNGKVIGEE LGYELVRYGRDREIKTEISVGDLVVISKRDPLKSDLVGIVVEKGKRFLIVAIETVPEWAI KGVRIDLYANDITEKRWMENLDNLRESGRKALELYLGLREPEESEPVEFQPFDKSLNASQ RGAIAKALGSGDFFLVHGPFGTGKTRILVELIRQEVARGHKVLATAESNVAVDNIVERLA DSGLKVVRIGHPSRVSKALHETTLAYLITQHDLYAELRELRVIGENLKEKRDIFTKPAPK YRRGLSDREILRLAEKGIGVPARLIREMAEWIRINQQVQKTEDDARKLEERIAREII QEADVVLITNASAGLEVVDYGEYDVAWIDEATQATIPSVIIPINRAKRFVLAGDHKQLPP TILSEKAKELSKTLFEGLIERYPEKSEMLTVQYRMNERLMEEPSREFYDGKIKARESVKN ITLADLGVSEPEFGNEWDEALKPENVLVFIDISKREDRFERQRRGSDSRENPLEAKLVTE TVEKLLEMGVKPDWIGVITPYDDQRDLISSMVGEDIEVKIVDGYQGREKETIVLSFVRSN RRGELGELTDLRRLNVSLTRAKRKLIAVGDSSTLSNHPTYRRFIEFVRERGIFIEIDGKK H >tr|C6A075|C6A075_THESMDNA helicase, UvrD/REPfamily OS = Thermcoccus sibaricus (strain MM739/DSM12597) GN = TSIB 2009 PE = 4SV = 1 (SEQ ID NO: 65) MIRVQIPAGAPKYGPVAUGQSARLISGRSGVRSPAGPPKALLKERFRELFIHKNPVITM HVKNYIAKLVDLVELEREAEIEAMREEMRRLKGYEREKVGRAILNLNGKIIGEEFGFKLV KYGRKEAFKTEIGVGDLVVISKGNPLASDINGTVVEKGSRFIVVALETVPSWAFRNVRID  LYANDITERRQLENLKKLSESGIRALKLILGKETPLKSSPEEFTPFDRNLNQSQKEAVSY ALGEEDFFLIHGPFGTGKTRTLVELIVQEVKRGNKILATAESNVAVDNLVERLWGKVKLV RIGHPSRVSVHLKESTLAEWESHERYRKVRELRNKAERLAVMRDQYKKPIPQMRRGLIN NQILKLAYRGRGSRGVPAKDIKQMAQWITLNEQIQKLYKFAEKIESEIIQEIIEDVDVVL STNSSAALEFIKDAEFDVAIIDEASQATIPSVLIPIAKARREVLAGDHKQLPPTILSEEA RALSETLFEKLIELYPFKAKMLEIQYRMNQLLMEFPSREFYNGKIKADGSVKDITLADLK VREPFFGEPWDSILKREEPLIFVDTSNRIDKWERQRKGSTSRENPLEALLVREIVERLLR MGIKKEWIGTITPYDDQVDSIRSIIQDDEIEIHTVDGYQGREKEIIILSLVRSNKKGELG FLMDLRRLNVSITRAKRKLVVIGDSETLVNHETYKRLIHFVKKYGRYIELGDTGIN >tr|W0I5I1|W0I5I1_9EURY DNA helicase, UvrD/REPfamily protein OS = Thermococcus paralvinellae GN = TES1_2001 PE = 4 SV = 1  (SEQ ID NO: 66) MNLIRYINHLKELVELEREAEIEAMREEMRKLIGYEREKVGRAVLGLNGKIIGEEFGYKL VKYGRKQEIKTEISVGDLVVISKGNPLASDLIGTVTEKGKRFLVVALETVPSWALRNVRI DLYANDITFKRQTENLDKLSESGKRALRFILGLEKPKESIDIEFKPFDEQLNESQKKAVG LALGSEDFFLIHGPFGTGKTRIVAEVILQEVKRGKKVLNFAESNVAVDNLVERLWGKVKL VRLGHPSRVSKHLKESTLAYQVEIHEKYKRVREFRNKAERLAMLRDQTKPITQWRRGLI DRQILRLAEKGIGARGIPARVIKSMAQWITFNEKVQRLYNEAKKIEEEIVKEIIRQADVV LSTNSSAALEFIKDIKFDVAVIDEASQATIPSVLIPIAKANKFILAGDHKQLPPTILSEE AKELSETLFEKLIELYPSKAKMLEIWRMNERLMEFPSKEPLINGKIKAYDGVKNITLLDL GVRVFSFGEPWDSILNLKEPLVFVDTSKHPEKWERQRKGSLSRENDLEAELVKEIVQKLL RMGIKPESIGVITPYDDQRDLISLLLENDEIEVKIVDGYQGREKEVIILSFVIRSNKKGEL GFLTDLRRLNVSLTRAKRKLIAIGDSETLSAHPTYKRFVEFVKEKGIFVQLNQYVSQTS >tr|B7AA42|B7AA42_THEAQ DNA helicase OS = Thermus aquaticus Y51M023 GN = TaqDRAFT_3809 PE = 4 SV = 1 (SEQ ID NO: 67) MGEAHPSEEALLSSLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVERVAYLIARRGVEPS EILAVTFINKAAEEMKARLKAMVRGAGELWVSTFHAAALRILRVYGERVGLKPGFVVYDE DDQTALLKEVLKELGLAAKPGPIKSLLDRAKNQGVPPEHLLLELPEFYAGLSRGRLUVL HRYQEALRAQGALDFGDILLYALKLLEEDGEVLKRVRKRARFIHVDEYQDINPVQYRFIR LLAGEEANLMAVGDPDQGIYSFRADIRNILDFTQDYPKARVYRLEDNYRSTEAILRFAN AVIVKNALRLEKTLRPVKKGGEPVRLFRAESARDEAREVAEEIARLGPPFDRVAVLYRTN AQSRLLEQALASRGIPARVVGGVGFFERAEVXDLLAYARLSLNPLDAYSLKRVLMTPPRG IGPATVEKVQAIARERGLPLFEALKVAALTLPRPEPLRAFLALMEELMDLAFGPARAFFR HLLLATDYPAYLKEAYPEDAEDRLENVEELLRAAKEAESLMDFLDKVALTARAEEPAEAE GRVALMTLHNAKGLEFPVVELVGVEEGLLPHRSSLSTQEGLEEERRLFYVGVTRAGERLY LSYAQEREIYGRLEPVRPSRFLEEVDEGLYEVYDPYRQSSRKPIPPPHRALPGAFRGGEK VVEPREGPGTVAAAGDEVIVHFEGVGLKRLSLKYADLRPA >tr|B7A5161B7A516 THEAQ DNA helicase OS = Thermus aquaticus Y51MC23 GN = TaqDRAFT_5093 PE = 4 SV = 1 (SEQ ID NO: 68) MRVYLASAGTGKTHALVEELKGLIQSGVPLRRIAALTFIRKAEELRGRAKRAVLALSAE DPRLKEAEREVEIGALFTTIEIGEMATEALRHTAPLLSLDPDFALLDTFLAEALFLEEARSLL YRKGLDGGLARALLHLYRKRTLAETLHPLPGAEGVFALYLEALEGYRRRLPAELSPSDLE ALALRILENPEALRRVVEREPHILLDEYQDTGPLQGRFFQGLKEAGARLVVVGDPKQSIY LERNARVEVEREALKQAEEVRYLSTTYRHAQAVAEFLNRETALFGEEGVRVRPHRQEVGR VEVHWVVGEGGLEEKRRAEAHLLLDRLMALREEGYAFSQMAVLVRSRSSLPPLEAAFRAR GVPYALGRGRSFFARPEVRDLYHALRLSLLEGPPGPEERLAILAFLRGPWVGLDLSEVEE ALKAQDPIPLLPEAVRAKLRALRALAGLPPLEALKRLSRDEAFLRRLSPRARVNLDALLL LAAMERFETLEALLEWLRLRAEDPEAAELPEGEEGWVLIVHGAKGLEWPVVALFDLSRG ENPKEEDLINGLGGEVALRGTPAYKEVRKALRKAQAEEARRLLYVALSRARDVLIVTGSA SGRPGPWVEALERLGLGPESQDPLVRRHPFKALPPLGDRPQTPPPPPLPAPYAHLAFPER PLPFVYSPSAFTKAKEPVPLAEALEKEALPEFYRALGILVHYAIARHLDPEDEGAMAGLL LQEVAFPFAEGEKRRLLEEVRDLLRRYRGMLGPSLPPLEAREEDHAELPLVLPLGGTVWY GILDRIYRVGGRWYLEDYKTDREVRPFAYRFQLAIYRRALLEAWGVFAEARLVYLRHGLV HPLDPEELERALKEGFPGMGPGEGGEKA >tr|B7A954|B7A954_THEAQ_DNA helicase OS = Thermus aquaticus Y51MC23 GN = TaqDRAFT_4764 PE = 4 SV = 1 (SEQ ID NO: 69) MKGLIGSSRLRVYGPPGTGKTTWLKNEVERLLRSGVPGEEIAVCAFSRAAFREFASRLAG QVPEENLGTIHSLAYRAIGRPPLALTKDALSDWNRRVPDTWRVTPRVDGRGADLLDVMDP YEDEDSRPPGDKLYDRVAYLRNTLAPMAAWSEEERAFFQAWKSWMNAKGLVDFPGMLEAA LAKPGGLGARFLLVDEAULTPLQLLLVEKWAQGARLALVGDDDQAIYGFMGADGASFLG VPVEDELVLGQSYRVPARVQRVAEAVIRRVQNRAPKRYAPRGDEGEVRLLWVPPEDPYHA VVDALERVNRGESVLFLATAKYLLEELKRELLRVGEPYANPYAPHRHSFNLFPQGARSAW EKARSELFPNRIAADVKAWTKHVSSKVEAVKGEEARRYLESFPDEEKVGDDHPIWNVERP EHRPHAVGRDVSWLLDHLLGNAPKTMRQSLMVAIKSPEAVLQGRARVWIGTIHSVKGGEA DWVYVWPGYTRKAAREHPDQLHRLFYVAAJTRARKGLVLMDQGKAPHGYVWPRVDEFWGEV WV >tr|H7GEQ71H7GEQ7_9DEINDNA helicase OS = Thermus sp. RL GN = RLTM0_2916 PE = 4 SV = 1 (SEQ ID NO: 70) MEANLYVAGAGIGKTYTLAERYLGFLEEGLSPLQVVAVTFTERAALELRHRVRQMVGERS LGHKERVLAELEAAPIGTLHALAARVCREFPEFAGVPADFQVMEDLEAALLLEAWLEEAL LEALQDPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRXLRR RMEELFHGERPEERYPGFPKGWRXEEPEVVPDLLAWAGEVKFNKKPWLEYKXDPALXRLL KLLGGVKEGFSPGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRAL EXEEVRAYYRGRERRLLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQSIYSFRRADP KVMERFQXEAAKEGLRVRRLEKSHRYHQGLADFHNRFFPPLLPGYGAVSAERKPEGEGPW VEHFQGDLEAQARFIAQEVGRILSEGFQVYDLGEKAYRPMSLRDVAVLGRTWRDLARVAE ALRRLEVPAVEAGGGNLLETRAFKDAYLALRFLGDPXDEEALVGLLRSPFFALTDGEVRR LAEARGEGETLWEVLEREGDLSAEAERARETLRGLLRRKALEAPSRLLQRLDGATGYTGV AARLPQGRRRVKDWEGILDLVRKLEVGSEDPFLVARHLRLLLRSGLSVERPPLEAGEAVT LLTVHGAKGIEWPVVEVLNVGGWNRLGSWKNNKTKPLFRPGLALVPPVLDEXGNPSALFH LAKRRVEEEEKQEENRLLYVAATRASERLYLLLSPDLSPDKGDLDPQTLIGAGSLEKGLE ATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPLAARDPEAARRRLLGEPEXEGGEA WXPXXPQETEEEVPGGAGVGRMTHALLERFEAXEDLEREGRAFLEESFPGAEGEEVEEAL RLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVGEDWVLDYKTDRGVDAXA YLLQVGVYALALGKPRALVADLREGKLYEGASQWEEKAEEVLRRLMGGEGQGRQPYPLA ATDPGHGAPG >tr|H7GE69|H7GH69_9DEINDNA helicase OS = Thermus sp. RL GN = RLTM07977 PE = 4 SV = 1 (SEQ ID NO: 71) MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVT FINKAAEEMRERLRGLVPGAGEVWVSTFRAAALRILRVYGERVGLRPGFVVYDEDDQTAL LKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEA LKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEE ANLMAYGDPDQGIYSFRAADIKNILDFIRDYPEARVYRLEENYRSTEAILRXANAVIVKN ALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAQSRLL EQALAGRGIPARVVGGVGEYERAEVKDLLAYARLALNPLDAVSLKRVINTPPRGIGPATW ARVQLLAQEKGLPPWEALKEAARTFXRAEPLRHEVALVEELQDLVEGPAEAFFRHLLEAT DYPTYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGKVALM TLHNAKGLEFPVVELVGVEEGLLPHRNSLSTLEGLEEERRLFYVGITRAQERLYLSHAEE REVYGRREPARPSRFLEEVEEGLYEVYDPYRXPKPXPPPHRPRPGAFRGGERVVHPREGP GTVVAAQGDEVIVHFEGXGLKRLSLKYAELXPA >tr|A0A0B0OSAG4|A0A0B0SAG4_9DEINDNA. helicase OS = Thermus sp. 2.9 GN = Q117 08170 PE = 4 SV = 1 (SEQ ID NO: 72) MDEALLSSLNEAQRQAVLHFQGPALVVAGAGSGKIRTVVHRVAYLIAHRGVYPTEILAVT FINKAAEEMRERLKGMVRGAGEVWVSTFHAAALRILRVITGERVGLKPGFVVYDEDDQTAL LKEVLKELGLSAKPGPIKALLDRAKNRGEPPEALLAELPEYYAGLSRRRLLDVFERYQEA LKAQGALDEGDILLYALRLLEEDQEVLARVRKRARFIHVDEYQDTNPVQYRFTKLLAGEE ANLMAVGDPDQGIYSFRAADIKNILQFLADFPGAKVYRLEENYRSTEAILRFANAVIVKN ALRLEKTLRPVKRGGEPVRLFRAKDAREEAREVAEEILRLGPPFDRIAVLYRTNAQSRLL EQALAGRGVGARVVGGVGFFERAEVKDLLAYARLALNPLDSVSLKRILNIPPRGIGPATV EKVARLAQEKGLPLFEALKRAELLPRPEPVRHEVALMEELMDLAFGPAEAFFRHLLQATD YPAYLREAYPEDHEDRLENVEELLRAAKEAESLLDFLDKVALTARAEEPAGAEGKVELMT LHNAKGLEFPVVELVGVEEGLLPHRNSLITTLEALEEERRLFYVGV7RAQERLYLSYAEER EVIGRLEATRPSRFLEEVEEGLYQEYDPYRSPRPVPPSHRPKPGAFKGGEKVVEPREGPG TVVAASGDEVIVHFEGVGLKRLSLKYADLRPA >tr|A0A084IL47|A0A084IL47_9GAMMATP-dependent DNA helicase Rep OS = Salinisphaera hydrothermalis C41138 GN = rep PE = 3 SV = 1  (SEQ ID NO: 73) MALPKLNPQQDAAMRYLDGPLLVLAGAGSGKTGVITRKIAHLIARGYDARRVVAVTFTNIK AAREMKQRASKLISADDARGLIVSTFESLGLQMIREEHAALGYKPRESIEDSEDADKVLA DLVGRDGDHRKATKAAISNWKSALIDPETATAQATGSDIPLARAYGEYQRRLKAYNAVDF DDLLALPVHLLSTDHEARERWQSRFRYLLVDEYQDTNAAQYEMMRLLAGARAAFTVVGDD DQSIYAWRGARPGNIADLSRDEPHLKVIKLEQNYRSVGNVLSAANQLIGASNQRAYEKTL WEAMGPGDRVRVIAAPDEAGEAERIASEISSHKLRLGTAYGDYAILYRGNEQSRAFEKAL RERDIPYRVSGGRSFFERSEIRDLVTYLKLMVNPDDDAAFLRIVNIPRREIGPATLEALG RYAGSRHISLFDAARGIGLAGGVGERSGRRLADEVDWLRNLTUSEGMTPRELVSQLIVD IDYRNWLRDTSANTKAARKRIENLDDFIGWLDRLDNAEDGKPVTLEDVVRRISLMDFANQ SEKDVENQVHLLTLHAAKGLEFDHVFLAGLEEGMLPHHACLEDDKIEEERRLLYVGITRA RKTLALTYARKRRRGGEESDSVPSRFLEELPADELDWPSATGTRSKAANAEQGRDQVAAL RAMLGASADS >tr|A0A0A2WMV1|A0A0A2WMV1_THEFIDNA helicase OS = Thermus filiformis GN = THFILI00990 PE = 4 SV = 1 (SEQ ID NO: 74) MPQVGFTDHEFKGLEALSREEQNRVREAVFAFMQDPKHPSFKLHRIEDIKTDREWSARVS KDLRLILYHHPEMGWIFAYVGHHDDAYRWAETHQAEVHPKLGLLQIFRVVEEVRVEPRKI KPLLPDYPDDYLLDLGVPPSYLKPLRLVETEDQLLGLIEGLPQDVQERLLDLAAGRPVTL PPKLAPSEETNEKHPLSRQHIHFIQNLDELRQALSYPWERWMVELHPAQREAVERVFQGPA RVTGPAGTGRTVVALHRAAALARRYPEEPLLLTTENRFLASRLRSGLQRLLGEVPPNITV ENLHSLARRLHEQHVGPVIKLVKEEDYGPWLLEAAQGLEYGKNFLLSEFAFADAWGLYTWE AYRGFPRTGRGVPLTARERLKLFGAFQKVWGRMENEGALTENGLLHRLRQRAEEGALPRF RAVVVDEAQDLGPAELLLVRALAQEAPDSLFFAIDPARIYKSPLSWQAIGLEVRGRSIR LKVNYRTTREIAKRAEAVLPKEVEGEMREVLSLLQGPEPEIRGFPTQEACQAELVRWLRW LLEQGVRPEEVAVLARVRKLAEGLAEGLRRAGIPVVLLSDQEDPGEGVRLGTVHSAKGLE FRAVALFaANRGLFPLESLLREAPSEADREALLAQERNLLYVAMSRARERLWVGYWDEGS PFLTP >tr|A0A0D0N7B7|A0A0D0N7B7_MEIRU DNAhelicaseOS = Meiothermus ruber GN = SY28 04645 PE = 4 SV = 1 (SEQ ID NO: 75) MSDLLSSLNPSQREAVLHFEGPALVVAGAGSGKTRYVVHRIAYLLRERRVYPAEILAVIF TNKAAGEMKERLEKMVGRSARDLWVTTFHAAAVRILRTYGEYVGLKPGEVIYDEDDQNTL LKEVLKELELEAKPGPFRSMIDRIKNRGAGLAEYMREAPDFIGGVPRDVAAEVYRRYQNS LRMQGALDENDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVORLTRLLAGER PNLMVVGDPDQSIYGFRNADINNILDFIKDYPGARVIRLEENYRSSSSILRVANAVIEKN ALRLEKVLRPTKPGGEPVRLYRAPNAREEAAFVAREIVKLGGYQQVAVLYRTNAQSRILE EHLRRANVPVRLVGAVGFFERREIKDLLAYGRVAVNPDDSINLRRIVNTPPRGIGATTVA RLVEHAQKTGITVFEAFRAAEQVISRPQQVQAFVRLLDELMEAAFESGPTAFFQRVLEQT GFREALKQEPDGEDRLQNVEELLRLAQDWEEEEGGSLADFLDSVALTAKAEEPQGDAPVE AVTLMTLHNAKGLEFPTVYLVGLEENLLPHRNSLHRLEDLEEERRLFYVGITRAQERLYL SYAEERETYGKREYTRPSRFLQDIPQDLLKEVGAFGDGETRVLSQARPEPKPRTQPAEFK GGEKVKHPKFGSGTVVAAMGGEVIVMFPGVGLKRLAVKFAGLERLE >tr|W2U4X3|W2U4X3_9DEINDNA helicase OS = Thermus sp. N1VIX2.A1 GN = TNMX_07060 PE = 4 SV = 1(SEQIDNO:76) MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRIV-VHRVAYLIAKRGVE PSEILAVTFINKAALEMRERLKRMVKGAGELWVSTEHSAALRILRVYGERVGLKPGFVVY DEDDQTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRILD VLKRYEEALKAQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDINPVQYRF TKLLAGEEANLMAVGDPDQGIYSFRAADIKNILEFTRDFPGAKVYRLEENYRSTEAILRF ANALIVNNALRLEKTLRPVKPGGEPVRLYRARDARDEAREVAEEILRLGPPFDRVAVLYR TNAQSRLLEQALASRGVPARVVGGVGFFERAEVXDLLAYARLSLNPLDGVSLKRVLNTPP RGIGPATVEKVEALAREKGLPLFEALRVAAEVLPRPAPLRHFLALMEELQELAFGPAEGF FRHLLEATDYPAYLREAYPEDHEDRLENVEELLRAAKEAEGLMEFLDKVALTARAEEPGE PAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHRSSLSTLEGLEFERRLFYVGVTRAQER LYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRASAKVSPSPAPSEARASKPKP GAYRGGEKVIHPREGQGTVVAAMGDEVTVHFEGVGLKRLSLKYADLRPVG >tr|H9ZQB5|H9ZQB5_THETHDNA helicase OS = Thermus thermophilus IL-18 GN = TtJL18 0620 PE = 4 SV = 1 (SEQ ID NO: 77) MSDALLAPLNEAQRQAVLHFEGPALVVAGAGS GKTRTVVERVAYLVARRGVEPSEILAVT FINKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTAL LKEVLKELALSARPGPIKALLDRAKNRGVGLEALLGELPEYYAGLSRGRLADVLVRYQEA LKAQGALDFGDILLYAIRLLKEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFIRLLAGEE ANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRLANAVIVKN ALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVIYRTNAQSRLL EQALAGRGIPARVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATW ARVQLLAQEKGIPPWEALKEAARTSSRVEPLRHEVALVEELQDLVEGPAEAFFRHLLEAT DYPTYLREAYPEDAEDRIENVEELLRAAKEAEDLQDFLDKVAITAKAEEPAEAEGKVAIM TLHNAKGLEFPVVFLVGVEEGLLPHRNSLSLEGLEEERRLFYVGITRAQERLYLSHAEE REVYGRREARESRFLEEVEELYEVYDPYRVPKPAPPPHRPRPGAFRGGERVVHPRFGP GTVVAAQGDEVTVHFEGFGLKRLSLKYAELRPA >tr|E8PM35|E8PM35_TRESS DNA helicase OS = Thermus scotoductus (strain. ATCC 700910/SA-01) GN = perAl PE = 4 SV = 1 (SEQ ID NO: 78) MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIAKRGVF PSEILAVTFTNKAAEEMRERLKRMVKGGGELWVSTFHSAALRILRVYGERVGLKPGFVVY DEDDQTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRLLD VLKRYEEALKAQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDTNPVQRF TKLLAGEEANLMAVGDPDQGIYSFRAADIKNILEFIRDFPGAKVYRLEENYRSTEATLRF ANALIVNNALRLEKTLRPVKPGGEPVRLYRARDARDEARFVAEEILRLGPPFDRVAVLYR TNAQSRLLEQTLASRGVPARVVGGVGFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPP RGIGPATVEKVEALARKGLPLFEALRVAAEVLPRPAPLRHFLALMEELQELAFGPAEGF FRHLLEATDYPAYLREAYPEDYEDRLENVEELLRAAKEAEGLMEFLDKVALTARAEEPGE PAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHRSSLSTLEGLEEERRLFYVGVTRAQER LYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRASAKVSPSPAPGEARASKPGA YRGGEKVIHPREGQGTVVAAMGDEVIVHFEGVGLKRLSLKYADLRPVG >tr|E8PL08|E8PL08_TRESS DNA helicase OS = Thermus scotoductus (strain ATCC 700910/SA-01) GN =perA2 PE = 4 SV = 1 (SEQ ID NO: 79) MLNPEQEAVANHFTGPALVIAGPGGKTRTVVHRIARLIRKGVDPETVTAV7FTKKAAGE MRERLVHLVGEETATKVFTATFHSLAYHVLKDIGTVRVLPAEQARKLIGEILEDLQAPKK LTAKVAQGAFSRVKNSGGGRRELIALYTDFSPYIERAWDAYEAYKEEKRLLDFDDLLHQA VHELSTDIDLQARWQHRARFLIVDEYQDINLVQFNLLRLLLTPEENLMAVGDPNQAIYAW RGADFRLILEFKKHFPNATVYKLHTNYRSHNGIVTAAKKVITHNTQREDLDLKALRNGDL PTLVQAQSREDEALAVAEVVKRHLDQGTPPEEIAILLRSLAYSRPIEATLRRYRIPYTIV GGLSFWNRKEVQLYLHLLQAASGNPESTVEVLASLVPGMGPKKARKALETGKYPKEAEEA LQLLQDLRAYTGERGEHLASAVQNTLHRHRKTLWPYLLELADGIEEAAWDRWANLEEAVS TLFAFAHHTPEGDLDTYLADILLQEEDPEDSGDGVKIMTLHASKGLEFAVVLLPFLVEGA. FPSWRSAQNPATLEEERRLFYVGLTRAKEHAYLSYHLVGERGATSPSRFARETPANLIHY NPTIGYQGKETDTLSKLAELF >tr|E4U8J8|E4U8J8 OCEP5 DNA helicase OS = Oceanithermus profundus (strain DSM14977/NBRC 100410/VKMB-2274/506) GN = Ocepr 1221 PE = 4 SV = 1 (SEQ ID NO: 80) MSARDLLSSLNEQQQAAVQHFLGPAIVIAGAGSGKTR7VVHRVAYLLAEREVYPAEVLAV TFINKLAGEMRERLSRMVGRAAGELWVSTFHSASLRILRRYGERIGLKPGFVVYDDDDQR VLLKEVLGSLGLEARPTYVRAVLDRIKNRMWSVDEFLAHADDWVGGLIKQQMAEVYARY0 QRLAENNAVDFNDLLLRTIELFERHPEALEAVRQRARFIHVDEYQDINPAQYRLTKLLAG DEANLMVVGDPDQSIYGFRNADIQNILGFERDYRGAVVYRLEANYRSTAAILRVANALIE RNQQRLEKTLRPVKPAGEPVRLYRAPDHREEAAFVAREVARLAGERALDDFAVIYRTNAQ SRVLEEAFRRLNLPARIVGGVGEYERREVKDVLAYARLAVNPADDVALRRVTNVPARGVG AASVGKLAAWAQAQGVSLLEAAHRAGELLAARQAAAVAKFTDLLTTLREAAEGTGPEAFL RLVLAETGYSEMLRREGDSEPRLENLEELLRAAEWEEEHGGSVAEFLDEIALTARAEEP NAAPEKSVILMTLHNAKGLEFPVVFVVGVEEGLLPHRSSLGSDAEIEEERRLLYVGITRA QERLYLTLSEERETWGQRERVRPSRFLEEIPEDFLKPVGPFGDAHEPAPAPLSSAPVNRA AKGSASGERGGEKVRHPRYGEGTVVATSGEGARQEVIVHFAEAGLKRLLVKYAGLERIE >tr|E4U4N5|E4U4N5_OCEP5 DNA. helicase OS = Oceanithermus profundus (strain DSM14977/NERC 100410/VKMB-2274/506) GN = Ocepr 1575 PE = 4 SV = 1 (SEQ ID NO: 81) MKVRIASAGTGKTYALTSRFTAALAEHPPYRLAAVTFTRSAAAELKARLRERLLAIAAGR FUSGAEDVPPEAVVRRAGALATEVLGATVTTIHGEFAELLRQNALALGLEPDFLRIDAS ESQQIFAEEARAYVYLNEEDDALAEVLGRLFAKRSLAAELRPQGEAAEALWAHFRAVLER YARRLGGEALGPADIELHAWRILERAGREEALAARIRSRLARVEVDEYQDTSPLQGRVFA ALEALGVEVEVVGDPKQSIYAFRNADVEVEREAMRRGEPLPPLVTSWRHDRALVRFLNRY VDWVAEERPEAFARAEAPPVEARPDAGPGRVRLQLVQGEARQDALRPYEADQLARWLQER HAEHAWRDMAVLVRSHSSVPLLVRALAAHGLPHVVVGGRGEYDLIEVRDLVHAARVALDP RGRFSLAAFLRGPFAGLDLGRVERVLAAEDPLAELERAPEVAERVDRLVRWVQLRPLD FFERMVRTPFLEGASYLERLEPPARANVDQLLFKLASRRYGRLEFLLRDLEDLRGSDEAG VPEGGFDAVRIYTMHGSKGLETNPVVAVEDLNRGUDGAEPFYVRPGSGEFAAEGDPDYPR FAAEWKERERQEAYRILYVALSRPRSRILLSLSVQLKPDGEGLRPKEWRRILGRTLIEEM NLAAWDALEVERLDAARLPAPKAPRRAADVDERLRAPVEPLARPPVYSPSALKAER PAPPELDDEGDVAVELEEPGVDPGLVARTVGILVHYAIGQDWGPERLQDLWNQEAVQRLT EPERTRVKTEVAQRLETYWRLLGTELPALDERDEDYAEFPLLLPIRTARLDTVWEGVIDR LYRVGDVWVLEDYKTDRELHPERYHFQLALYRRAVAAAWGIEPEARLVYLRFGEVVPLEA QLLEEAFERGTREAEEV >tr|E4TJAI1|E4UAI1_OCEP5 DNA heiicase OS = Oceanithermus profundus (strain DSM14977/NBRC 100410/VIMB-2274/506) GN = Ocepr_2312 PE = 4 SV = 1 (SEQ ID NO: 82) MKVIVASA = KTTRLTQRYLEHLEQHPPQRVAAVTFTNKAAAELRERIFEALGRGSFYD FTPSPALAERLADYQVRVLEAPIGTIHSFFGYLLRLTAPMLGLDPHFEVTDPATARAWFL EEVRNLAIIEGAEVDETVITALVELFKRRSISEAFEGTGDASRSLVAGFKKVYARNLTRL GGRYLDPSEIERRALALIRHPEALERVRSRLGVVLVDEYQDTAPIQARVFEALEEAGVPI EVVGDPKQSIYAFRDADVEGFREAERRARENGNVETLIVSYRHPPALADFLNAFTSAEAA LGKAFTAEEAPEVKPGREGDARVELITVIPGDGKATLDALRNGEARLLARELRRLHDEEG YDYGQMLVLERRRHQLPPLLRALRGAGLPFAVVGLRGLYEEPEVRELYHALRLATGEAPR DSLAVELSGPFGGLTLGQVREVLAQDAPESYLTLHEPEAAERLLRLRADAEKMRPAEALT RLIEAPTAKGPPFLDLLELEMADTVLYVLGRIEHTRTYPEAVATLESFRSGGEEEASLAR LGGDAVRVMSAHAAKGLQAPVVVIFDADRTFNGNSDELVIEPRIGRVALNGEDAYESIAQ ALKARKEGEDHRLIYVALSRSSERLIVSAAVKEPRKGSWLHHLTEVLNLGSKFEHRNVTL AEIALEEPIEQEAATLPVDPELATPLPPAPPAVSSPTALKAERELEVPDPEEAWPADPEA RLLGRIVGILVHEGIQRDWDPDDPEVLLALEGEQVLEEVPADRRPAVIEEVAILLRVYRT LLGSAIPSLEEREVDLAELPLVYPLGATAWEGVIDRLYRVGDVWYLEDYKTDREVHPERY HSQLALYREAVRKHWGIEPEVRLVYLRTGQVVPLDAAALKEGLASYTGG >tr|E4UAI8|E4UAI8_CEPS DNA helicase OS = Oceanithermus profundus (strain DSM14977/NBRC 100410/VIMB-2274/506) GN = Ocepr 2319 PE = 4 SV = 1 (SEGID NO: 83) MNEHERVIAHEVGPAAVVAGAGSGKTRAATLRAARLARTGERVGLVTFTASAAEEMRQRV LAEDVPAKHVWAGTFHSLAFQILRQFPEAGGYEGFPEVLTPNDELRLFRRLWAELLDQDL DAELRRKLVKALGFFRKARAEEALEGWAARAGESLELDAEMLEALMISFQLRKREAGLAS FDDLIEGASRALGDKDVRKWADRRFPFLIVDEYQDTSRAQETFLAALMPGEAPNLMVIGD PNQAIYGWRGAGSRTFERFQARYPQAVLYPLRKNYRSTRAVLRIAERAIARIYRSGUAY YRLEGVKEEGEPPVLLTPPNAAAEATDVAREVARAVASGVPPEEIAVLARSSMQLAGVED RLARLGVATRLLGGIRLSERREVKILVQLLKAAWSLHERALVDFIEEAVPGLGERTLTRV EHAARPYNLVDRIMNDGAFVRGFSTRVQQGLFMTRTLLQLARATFEGVTGEAFAERFREF AQDLYGELLPGYLARIGKQGPNEEARRRHLERFVATVEAFAREEAEGGLDDLLARLAFLE QQDGPAVTLGTVHAVKGLEFEVVFIVGMVEGAFPILADDSDPEEERRLFYVAAJTRAKRRL YLSAPTYGPRGKILUSRYLEEALDEGLVRLQKVRPAA. >tr|E4UAI4|E4UAI4_OCEP5 AAA ATPase OS = Oceanithermus profundus (strain DSM14977/NBRC 100410/VIB-2274/506) GN = Ocepr 2315 PE = 4 SV = 1 (SEQ ID NO: 84) MVSEGRWKIERVVYLKDGFAV-VAVRNEAGERHTAVGEMPTPVEGTWVRMETEHTVHPRYG PRLRVVRFLGLAPPPSKELAKIEGYLKLGFSEFLASWLAARFGSRPERAFDKPQELLVPG VPREVLRRVFPRLERLLGGLIDLLGEGHTAAPLFLLAERSGLGKEEIQELAREARKQRLI VEEQGRYGLVQPYRTERSIADGLLFRLKPGRGLRLIPPAGHGLSDEQARIFKLVRENRVV VLIGGPGSGKITTIATLLAAPELHRMREGIAAPTGKAARRIAEVARLPAETIHRLLGLGE ARRPLYHARNPLPYDLLVIDETSMLDAEIAAFLVDALAPSTSWIFVGDPDQLPPVGPGQF LRDLMTRVATLRLTQIFRQAQDSPIVNGAYALREGRMPLADGERLRLLPFEEEAAQTTLR ILLDELQRLEQIVGERPQNLVPGNRGPLGVRRLSPFLQQQLNPGGKPLGPIGWGMEAREG DPAVWIHNDYELGIMNGEVGVLRGGGSLGLTFETPTDRFAIPGNKRSRLVLAYAMTVHRS QGSEWPAVITILPKAHMALLSRELVYTALTRSKQYHTLLFHPEALYRARAVQASRRYTWL DVLLRG >tr|K7QW32|K7QW32_THEOS DNA helicase OS = Thermus oshimai JL-2 GN = Theos_1787 PE = 4 SV = 1 (SEQ ID NO: 85) MTAPGHPDALLAPLNPAQQEAVLHFQGPALVVAGAGSGKTRIVVHRVAYLMAHRGVYPGE ILAVTFTNKAAEEMKGRLKALVPGAGELWVATFHSAALRILRVYGEAIGLKPGFVVYDEA DQEALLKEVLKELGLSAKPGPLKALLDRAKNRGEAWEALEIPDYYAGLPKGKVLDVLRRY QEALRAQGALDFGDILVYALRLLEENPEVLAKVRKRARFIHVDEYQDTSPVQYRFARLLA GEEANLMAVGDPDQGIYSFRAADIRNILDFTRDFPGARVIRLEENYRSTEAILRFANAY-1 QKNRLRLEKTLRPVKPGGEPVRVIAAPEAREEARFVAEEIFRLGPPYERFAVLYRTNAQS RLLEQALAAKGIPYRVVGGVGFEERAEVKDLLAYARLSLNPEDGVSLKRVLNIPPRGIGP ATLARIEALAQAEGVPLLGAIRLGAERFPKPEPLRAFLALLDFLADLAFGPPEAFFRELL SATDYLQYLKEHHPEDAEDRLENVEELLRAAKEAULQEFLDRVALTARADQDGGRGVAL MTLHNAKGLEFPVVFLVGVEEGLLPHQSSLSTLEGLEEERRLFYVGVTRAQDRLYLSYAR EREVYGRREPRRMSRFLEEVPEGLYLPHDPYRQGAUKPAPRAQGAFRGGEKVVHPREGP GTTVVAASGDEVIVHFEGVGLKRLSLKYADLRPA >tr|K7QWX5|K7QWX5 THEOS DNA helicase OS = Thermus oshimai JL-2 GN = Theos_2419 PE = 4 SV = 1 (SEGID NO: 86) MASSLSKAELVPTPEQEKALHLYRSRUFKLVAVAGSGKITTLRLMAESFPRRHIAYLAF NRAMKEEARRKFPPNTRVFTLHALAYRRTVPGTPYEAKFRLGNGQVRPVHVRERLQVDPL LAYVVRSGLERFIRSGDPEPLPRHLPRDWRKIVEARGPSGFAEVERAVKGVALLWKAMRD PHDPFPLSHDGYVRIWREEGAGGDPPAGWILVDEAQDLDPNELIVISGWRGKAQQVFVGD PRQQIYGWRGAVNAMGEIDLPESPLIWSFREGEPLASFVQAVTARQTQGLVPLVGRAGWA TEVHVNLEPTPPFTILTRSNLGLVTALLEGAQLFSLQKEEAHVVGGVEFLVWLLTDLQAI KEGGERPRPHPELLGISKWEEVESLAEYSIVLNRLLRLAKEYDLEALAHKIAQLHGPEEG AKLVLSTAHKAKGREWDRVLLWEDFYWVAAYRWFFPNTAPPPSEPSPEFLEEENIFYVAM TRARLGLHISLPEALAEEEAKRILDRLSQGVPSGEDRGEDERGETLPAPFTGPTPVSPKE ATFPLPSLYDRLLSEALNGGRDPLLHLLRDDLARLSALSPTPLPPEVAQALWERARPEEA LGAIREGLGAMWREDPYELLRAINALALLGGRNPRKLAKILGDRFPGGEEAEDLLFVARA RKRELMGRSLAEFWRGLGASVRHPLLKAYARAIRS >tr|K7QTS9|K7QTS9_THEOS DNA helicase OS = Thermus oshimai JL-2 GN = Theos_0356 PE = 4 SV = 1 (SEQ ID NO: 87) MRLYVASAGTGKTETLMGELKALLEGGVPLRRVAAVSFIRKSAEELRLRVRRLLEAHREA FWAREALREVEGALFTTLHGEMAEALRHTAPFLGLDPDFRVMDGFLAQALFLEEARSLLF LEGHPEAPELLELLEAGTGEKRSLAEAFTPLPGAEGLLALYERVLARYRARTQEVLGPGDL EAKALLLLRHPEALGRVAERFSHLLVDEFQDVNPLQGRFLRALEEAGVRVVAVGDPKQSI YLFRNARVEVFLRARAAEEVRALSRTHRHAKQVVELLNRETTREFRAEEGNRVEGVREA EGRVEVHWVLGKLEEARRAEARLLAQRLLALRAEGIPFGEMAVLVRARTSLPPLEKALRA AGVPFVRGRGQSFEARPEVRDLYHALRLALAERPYALEDRLSLLAFLRSPFLGLDLSELE EALRAEDPWPLLPKGVQEALEGLRALALLPPLEALRRLARDEGFLRRISRRARANIDILL LLAAGAREPTLEDLLLWLALRAKDPESVELPEGGGGVTLLTVHGAKGLEWPVVALYDVSR GPSERPPPLLVDEEGRVALKGTEAYRALLKEAERAEREEALRLLYVALSRARDLLLITGS TSQRPGPWAEALQALGLGPDAQDPWVETHPLEAIPPLPPIPQAPQDPRPAPYTPWRGEPR ARPPVYSPSAHLKAEAEPLEVLGEGEALPEWARAVGTLVHYAIARHLDPEDEGAMGGLLR QEVALAFGEGEREALLEEVRALLRAYRSLLSGALPPLEARAEDHAELPLLLPHKGIVWYG VLDRLYRVGDRWYIDDYIKTDQKVRPEAYRFQLALYRKAVLEAWGVEAEARLVYLRHRQVV PLSPAELEAALEGL >tr|D1AF88|D1AF88_THECD DNA helicase OS = Thermomonospora curvata (strain ATCC 19995/DSM43183/JCM3096/NCIMB 10081) GN = Tcur_4104 PE = 4 SV = 1 (SEQ ID NO: 88) MSSSQVTGRPTIVKDAEIAVEQRRVDQAHARLEEMRAEAQAMIEEGYRQALAGIKGSLVD RDAMVYQAALRVQALNVADDGLVEGRLDLADGQTRYIGRIGVRIRDHEPMWIDWRAPAAE AFYRATPEDPQGVVRRRVLHTRGRTVVDLEDDLLDPSAADSLTIVGDGAFIASLARTREG TMRDIVATIQREQDEVIRAPADGTVLVRGAPGIGKTAVALHRVAYLLFRERRREGSRGVL VVGPNRRETAYIERVLPSLGEGSATLRSLGDLVEGVSATVHDPPELAALKGSAAKAPVLR RAVTDHPPGAPDKLRVVHGGVVVELGRPQLDKLRTSLHRRSTGSVNASRRRVAEALLDAL WERYVHIGGTEPEPDEPVQGELALWEGILAEGGLAPLDEQDRPSSPADRTSREAFVKNVR EQRAFTDELTAWWPIRRPLDVLRSLGDAARLRRAAGRDLDRAQVELLAASWRRALAGDPP TLSYQDIALLDEIDALLGPPPQPSRATAREEDPYVVDGIDILTGEVVADEDWEPGLQELT TTIERLERARRVDDEVADVRPEYAHIVVDEAQDLSPNQWRMLGRRGRQATWTIVEDPAQS AWEDLEEARKAMEAALDGPAARRGRSRRPRRRPRHEYELTTNYRNTTEIAAVSARVLRLA LPEARPARAVRSSGHRPVIDLVPEEELQAAARRAVRILLEQVEGTIGVIVPLPGDAWGES DRRALSAGFPERVQVLDVLEAKGLEFDAAVICAPETIAAQSPRGLRVLYVAVSRATQRLT VLTADPVWRRRLAGGESAR >tr|F8A884|F8A884 THEID DNA helicase OS = Thermodesulfatator indicus (strain. DSM15286/JON11887/0IR29812) GN = Thein 0607 PE = 4 SV = 1 (SEQ ID NO: 89) NTSISLDQYQEQAVKAKGNTLVVAGPGAGKIRVLLAKAIHLLEQGIDPEKVLILIFTIKT TQELKERLASIGIKGVKVDTFHALAYDLLKAKGIKPRLATEEELKALARDLSKRKGLSLK DERKALDKGENHYRSLWEEALKLHGLYDFSLLLKEATGHYLQQEKVYLLIDEFQDLNPEL TSFLKTFTKAEFFLVGDPAQATYGERGACPQVIKEFVDYLAPQIYFLKKSYRVPEKVLNE AETLRETQGFPLEPLEAVQKGGNRLGLSENKPFNEAKGVAKLVSELLGGLQMEASQRGLA PPEIAILSRVRILLNPIKEAFIKEGIPFQVPSENLKEEISATESLSDIAKSIKSLKELEA YLAEGPSSVKEAWLESQSLEGFLFRLEMLKTFASISIRKDGVPLLTIHEAKGLEFKVVIL VGAEDGLLPFTLLEDYDLAEEKRVAYVAVTRAQESFYFTWKIGRFLYGHKLSGKVSPFF ETLPIKEKSSKTKPKARQKKLFG >tr|A0A087LEB0|A0A087LEB0_GEOSE Uncharacterized protein OS = Geobacillus stearothermophjius GN = GT94_17890 PE = 4 SV = 1  (SEQ ID NO: 90) NTISVIDELLEKNKQNNNKTAKDAVEAQLIAYAKKEVKKLUIRPHPYFGRLDFEDEFGR ETIYIGKKGLEKDGELIVVDWRIDLGRLYNAYQGVQKTFQIGKENRPVTIHGKRGIVIKN GKVIKVIDIGKSEIIENDNGEKVKYNDDYLKEILTNTEEAHRLRDITASIQAEQDETIRL PLKDTIIVQGAAGSGKSTIALHRISYLLYWHEQVXPKDILILAPNEIFLSYIKDIVPEI EIEGIEQRTFYDWASTYFTDVHDIPDLHEQYVHIYGSTEKEDLIKIAKYKGSLRFKKLLD DEVEYIGNTNIPHGDVVIESGVILSKEEIHQFYHAKEHLPLNVRNKEVKEFIINWRNEQI NIRKQQIEDEFEEAYRKWVVTLPEGEERKAVYEALEKAKQLRMKIFQEKNQHEISLIVKK MENIPALLNYKSVFQKKVFEKFHPDIDEELLSLLLKNGRQIKQERFMYEDIAPLIYLDAK INGKKLWEHIVIDEAQDYSPFQLAINKDYAKSMTILGDIAQGIFSFYGLDRWEEIESYV FKEKEFKRLHLQTSYRSTKQINDLANRVLLNSNIDEPLVIPVNRPGDVPTIKKVESIGEL YDEIVNSIRIFEEKGYKKIAILTASKQGAIDTYDQLNRRQITQNEVITEGHQALKEKIVI IPSYLVKGLEFDAVIIEDVSDETEKDETQHAKMLYNSITRAHHDLHLFYRGNISPLLEER DPSAPPKPRKSFADWLITDINDPYVEPQVEAVKRVKKEDMIRLFDDEEEEFVEEAFEDDR ERYYDFHAWLKVWRRWAEMRKQLDEKS >tr|B5Y6N2|B5Y6N2_COPPD DNA helicase OS = Coprothermobacter proteolyticus (strain ATCC 35245/DSM5265/DT) GN = perA PE = 4 SV = 1 (SEQ ID NO: 91) MALPQENLIPPSPSHNHLTLSLRSHIGGFFIYNEDVDSVDLSKLNEAQKQAVTAPPKPLA IIAGPGSGKTRVLTYRALFAVKEWHLPPERILAITFINKADELKERLGRLIPEGDRIFA ATMHSFAARMLRYFAPYAGISQNFVIYDDDDSKGLIEDILKQMNFIDTKRFRPNDVLNHIS AAKARMFDCNTFPEFIRQRYGSWGITYFDTVHQVFMTITERLKEQSQALDFDDLIMVLAQRN EDRPELRENTAGLFDLVMVDEFQDINFAQYQNLLYNTNPHYSGMNNVTIVGDPDQSIYGF RAAETYNIKRFIDDYNPEVVELDLNIRSNRTIVDSASALINDSPSALFERKLESIKGAGN KLILRRPFDDADAAITAAFEVQRLHKNGIPYEEIAVLMRTRALTARVEREFATRNIQYEI IGGVPFFARRETKDILAYLRLSRNAMDRVSLKR:ILTMKKRGFGTABLEKLENFAEENKLT LLEAMKAAVESLLFKKLSMNDYLESLYTLIQTIQEIAEPSQAIYLVNEUNLLDHFRSIS KSEEEYIERTENVKQLISIAEESADMDDFLQRSALGTRENNGGVEGVAISTVHGVKGLEF QAVILYYVTDGFFPHSLSVITAEKEEERRLLYVAMTRAKEHLIFYVPYKQPWGNGFEQMA RPSPFLRSIPKELWDGKPNEIESLYAPYBPQQKWSE >tr|D7BJL0|D7BJL0_MEISD DNA helicase OS = Neiothermus sjivanus (strain. ATCC 700542/DSM9946/VI-R2) GN = Mesil 3574 PE = 4 SV = 1 (SEQ ID NO: 92) MNDPIRHKEGPALVFAGAGAGKTRILTQRVKWLVEEGEDPYSITLVTFINKAAGEMKERI ARLVEAPLAEAVWVGTFHRFCLQSLQVYGREIGLEKVAVLDSAAQRKLAERIIAGLFPAK PPRGFIPMAALGAVSRAANSGINDDIQLATMYADLTEKIVNERWAYEEAKKGLGALDYDDL LLRGVRLLKLSEGAARMVRRRAAYLMVDEFQDINGVQLELVRAIAPGISPNLMVVGDPDR SIYGWRGANYRTILEFRQHYPGAAVYGLYTNYRSQAGVVEVANRIIAQNATRKPEMQEAH LPQSEEPFLLVAKNRWEEAHFVAQAVEFYRGQGIALEENAVLNRANFLSRDLEQALRLRG IPYQFTGGRSFFERREIQLGMAVLKVLANPKDSLAVAALVEEMVEGAGPLGIQKVLEAAK AANLSPLEAFRNPAMVKGLRGKEVQAEANRLAEVLQDQVARLAAEAPEYHALLKEILDRL STEAWIDRLGEESEQVYERKANLDRLLQGMQEWQEVNPGAPLQDLVGILLLEAGDIPAEE GQGVHLNIVHASKGMEFRVVEVIGLNEGLFPLSKASSSFEGLEEERRLNYVAV7RAKEVL HLSYAADGVVSRFAQEARVPVEEYDPRIGWSGRQNQQALKALLEIA >tr|E8MZN5|E8MZN5_ANATU DNA helicase OS = Anaerolinea thermophila (strain DSM14523/JON11388/NBRC 100420/UNI-1) GN = perA PE = 4 SV = 1 (SEQ ID NO: 93) MDSLEHLNPQQRAAVIASAGPVLVLAGPGSGKTRVITFRIGYLLSQLGVAPHHILAVIFT NKAARENQSRVEKLLGHSLQGMWLGTFHAICARdLRREQQYLPLDANEVIEDEDDQQALT KRALRDLNLDEKLYRPTSVHAAISNAKNNLILPEDYPTATYRDEVVARVYKRYQELLVSS NAVDFDDLLLYAWKLLNEFSTVREQYARRFEHILVDEFUTNLAQYELVKLLASYHRNLF VVGDEDQSIYRWRGADYRNVLRFEEDFPDRQKILLEQNYRSTQRVLDAAQAVINRNRNRI PKRLKSTPEHGEGEKLVLYEAVDDYGEAAFVVDTIQQLVAGGKARPGDFAIMYRTNAQSR LLEEAFLRAGVPYRLVGANRFYGRREVKDMIAYLRLVQNPADEASLGRWINVPPRGIGDK SQLALQMEAQRTGRSAGLILMELGREGKDSPHWQAIGRNAELLADEGSLLGEWHRIKDEI SLPSLFQRILNDLAYREYTDDNTEEGQSRWENVQELLRIAYEYEEKGLTAFLENLALVSD QDTLPENVEAPTLLTLHAAKGLEFPIVFITGLDDGLIPHNRSLDDPEANAEERRLFYVGL TRAKKRVYLVRA_AQRSTYGSFQDSIPSRFLKDIPADLIQUGRGRRNGRSWQSESRRSWD DNYAGTWGSRPERAKPSHAPILQPRFKPGMRVKHPSWGEGLVVDSRIQDEDETVDIFFDS VGFKRVIASIANLEILS >tr|L0INW7|L0INW7_THEIRATP-dependent exoDNAse (Exonuclease V), alpha subunit/helicase superfamily Imember OS = Thermoanaerobacterium thermosaccharolyticum M0795 GN = Thethe 02902 PE = 4 SV = 1 (SEQ ID NO: 94) MDINGQIIKLNRNKTQGTLKLINGQKIKFKINSDSVKPIFLYEYYKFKGNMIEDTLIIDD IYGIANDININDFIELFPSVAHDKINNICNRENVLHVGNLIDLINDENFITVVNDTIGEE KATIFLSNLQKIKDRQEYIDVWDIIKKINPTEDINVPIKIVNALKYRASMNNITVSQLIK ESPWIIEQLDIFDSITERKKIAENIATHYGLSNDSNKAVISYAIAMTNNYIQQGHSYIPY YILVSRVSNSLKLDENKVNDTLKFLPNDNKSGYLIRDNKYKDFIENFYNSDKKIGYSVYL PKIEHMEKYIADIISSILKKKSTINKIELQKNLKLYRSENKLIFSKEQEFAIFSISDNKI TVITGGAGTGKITVIKAIIDLVNKMGYIPVVLAPTGIASQRVAPNVGSTIHKYARIFDTY DPVEDEIEENKENNSGKVIIVDEMSMITVPVFAKLLSVILDADSFIFVGDPNQLPPIGAG GVFEALIELGNKNINNINTVVLNQSFRSKNSIVXNAQNILEDKPIYEDDNLNIIEAKSWN KIADEVVNLIRKILDNGVQYSDIMVLSSKRGEGKNGVELLNERIRKEIENNKGKYAVGDI VITIRNDYDNKSSYFRSKELKKYINSIRHEERPTIENGTVGVIKDISDNEVIIEYNTPMP VEAKYNMEFLDWYIEYGFAITVHKAQGGQAKYIIFASDEPRNISREMLYTAITRCKNGKV FLIGGENEDWKIKKEHSFVLSKLKYRILDNIHQOEKESKINSKIVLINQ >tr|D3PR99|D3PR99_MEIRD DNA helicase OS = Meiothermus ruber (strain ATCC 35948/DSM1279/VKMB-1258/21) GN = K649 05745 PE = 4 SV = 1 (SEQ ID NO: 95) MSDLLSSLNPSQQEAVLHFEGPALVVAGAGSGKTRIVVHRIAYLLRERRVYPAEILAVTF TNKLAGEMKERLEKMVGRPARDLWVSTFHAAAVRILRTYGEYVGLRPGFVIYDEDDQNTL LKEVLKELELEAKPGPFRAMIDRIKNRGAGLAEYMREAPDFIGGVPKDAAAEVYRKYQSG LRMQGALDENDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVQYKLTRLLAGER PNLMVVGDPDQSIYGERSADINNILDFIKDYPGARVIRLEENYRSSSSILRVANAVIEKN ALRLEKVLRPTRPGGEPVRLYRAPNAREEAAFVAREIVKLGNEXIAVLYRINAQSRLLE FHLRRANVPVRIVGAVGFFERREIKDLLAYGRVAVNPADSINLRRIVNIPPRGIGATIVS RLVEHAQKTGTIVFEAFRVAEQVISRPQQVQAFVRLLDELIEAAFESGPTAFFQRVLEQT GFREALKQEPDGEDRLQNVEELLRPAQDWEEEEGGSLSDFLDSVALTAKAEEPQGDAPAE AVTLMILHNAKGLEFPTVFLVGLEENLLPHRNSLHRLEDLEEERRLFYVGITRAQERLYL SYAEERETYGKREYIRPSRFLEDIPQDLLKEVGAFGDSEVRVLPQARPEPKPRIQLAEFK GGEKVRHPKEGSGIVVAAMGGEVIVMFPGVGLKRLAVKFAGLERLE >tr|D3PLL2|D3PLL2_MEIRD DNA helicase OS = Meiothermus ruber (strain ATCC 35948/DSM1279/VIMB-1258/21) GN = K649 10770 PE = 4 SV = 1 (SEQ ID NO: 96) MKVRVASAGTGKIASLVLRYLELIAKGIPLRRIAGVTFTRKAADELRVRVAAAIEEVIQT GRELSEVASGGSRAAFQEAAREIAGATLSTIHGEMAQCLRLAAPILHLDPDFSMLGDWEA QAIFFEEWQTLRYLAQDAHHPLEGLVSDFLTEPLLHLFSRRSQAEVFEPAAGEANQHLLQ VYQTVYAAYEARLGANLLSPSELERKALFLARNDRAMKRVLERVRVLLVDEYQDVNPVQG AFFLALEQARLPIEIVGDPKQSIYAFRNADVSVERKALREGKSEPPLIHSYRHSRVLVRF LNGLIGYLAKEGLGEGLEEAPPVEGVRPEQGRLEVHWVVGELPLEELRKQEARVLAGRLA ALRGPIEYSQMAVLVRSYGSVRFLEEALAEAQIPYVLLQGRGYYERQEVRDLYHALRAAL DPRGLSLAVFLRSPFGQHTEAGPLKPLELPQIEGVIRADDPLGRLAQHWPSVYERLRQIQ AQVRLMAPLEVLKFLIRAPLMDGRPYHDFLEPRARENVDALLFYFAPRPPQNLEGLLERL ELLSRQADAGDVPQSGEGVQILIVHQAKGLEWPLVAVEDLGRMNVHRPQPLYIGQGPNGG DGGRLRRWVALPETPQFEAFRQQVKLUEEESYRLLYVAASRARDILLLTASASHGQPEG WGKVLEAMNLGPASKPYHRPDFHLQIWPYQPAPPVRVLSQPAPLQPSPWVDARFEPEPFP PLFSPSALKRLEAEPLPLPDPEEGEAVPGRARAIGILVHYAIGQNWRPDNPQHLANLEAQ EVMFPFGPDERRGIMAEVQALLEHYQELLGRALPWPRDEDYPEFAVALPLGSTVWQGVID  RLYRVGQQWYLEDYKTDQEMRPERYLVQLGIYLAAIRQAWQIEPEVRLVYLRFGWVERLD KAILEAALGEIMPKGEGIRR >tr|Q9RTI9|0RTI9_DEIRA DNA helicase OS = Deinococcus radiodurans (strain ATCC 13939/DSM20539/JON16871LMG 4051/NBRC 15346/NCIMB 9279/R1/VKMB-1422) GN = DR 1775 PE = 4 SV = 1 (SEQ ID NO: 97) MTSSAGPDLLQALNPTQAQAADHFTGPALVIAGAGSGKIRTLIYRIAHLIGHYGVHPGEI LAVTFINKAAAEMRERAGHLVPGAGDLWMSTEHSAGVRILRTYGEHIGLRRGEVIYDDDD QLDIIKEVMGSIPGIGAETURVIRGIIDRAKSNLWTPDDLDRSREPFISGLPRDAAAEA YRRYEVRKKGQNAIDEGDLITETVRLFKEVPGVLDKVQNKAKFITHVDEYQDTNRAQYELT RLLASRDRNLLVVGDPDQSIYKFRGADIQNILDFQKDYPDAKVYMLEHNYRSSARVLEAA NKLIENNTERLDKILKPVKEAGQPVTFHRATDHRAEGDYVADWLTRLHGEGRAWSEMAIL YRTNAQSRVIEESLRRVQIPARIVGGVGFYDRREIRDILAYARLALNPADDVALRRIIGR PRRGIGDTALQKLMEWARTHHTSVLTACANAAEQNILDRGAHKATEFAGLMEAMSEAADN YEPAAFLRFVMETSGYLDLLRQEGQEGQVRLENLEELVSAAEEWSQDEANVGGSIADFLD DAALLSSVDDMRTKAENKGAPEDAVTLMILHNAKGLEFPVVFIVGVEQGLLPSKGAIAEG PSGIEEERRIFYVGITRAMERILMTAAQNRMUGKINAAEDSAFLEDIEGLFDTVDPYGQ PIEYRAKTWKQYRPTVRAATTAVKNTSPLTAELAYRGGEQVKHPKFGEGQVLAVAGVGER QEVTVHFASAGIKKLMVKFANLTKL >tr|M1E5C5|M1E5C5_9FIRM DNA helicase OS = Thermodesulfobium narugense DSM14796 GN = Thena 1375 PE = 4 SV = 1 (SEQ ID NO: 98) MDLNLNEDQKRAVYSDSRALLIVAGAGTGKTRVLTTRAARLIKENPDARYLLLIFTKKAA REMTTRVRELIEEDTKNRLYSGTEHSFCSNIIRRRSERVGLINDFVIIDESDSLDLMKKV FSRIYSKEKIDSLIFKPKDILSLYSYARNNNQDFIEIVQRKYKYVNFEDIKKIISLYELN KKERNYLDFDDLLMYGLLAIKTLEKSPFDEVLVDEFUTNQIQAEMLYYFYDLGSRISAV GDDAQSIYSFRGAYYENMENYIKRLDAEKliLSSNIRSTQQILDIANSIIQSSYSSIKKE LVANVRLKENVKPKLVIVSDDWEEARYVAREMQKFGEKGLKVAALYRAAYIGRNLESQLN SMGIKYSFYGGQKLTESAEAKDFMSFLRVEVNPKDEIALIRTLKMFPGIGEKKAFKIKDA VISGDNLKKALSKEKNLEELNIFFDKLFKITDWHDLLELVFDFYKDIMNRLYPENYEERE EDLIKEMDMSSNYDNLVEYLEAFTLDPVEKSEFDNNNVILSTIHSAKGLEFDVVFLLSVI ESVYPHFRAQSTDEIEEERRLFYVAITRAKQRLIFTFPRHSKKSRGYFAKNTISPFLREK DNYLEVFIAR >tr|OSIE7|Q5SIE7_THET8 DNA helicase OS = Thermus thermophilus (strain. HB8/ATCC 27634/DSM579) GN = T1HA.1427 PE = 4 SV = 1 (SEQ  ID NO: 99) MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVT FINKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVITGERVGLRPGFVVYDEDDQTAL LKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEA LKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEE ANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKN ALRLEKALRPVKRGGEPVRLYRAEDAREEAREVAFETARLGPPWDRYAVLYRTNAQSRLL EQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNIPPRGIGPATW ARVQLLAQEKGLPPWEALKEAARTFSRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEAT DYPAYLREAYPEDAEDRLENVEELLRATIKEAEDLQDFLDRVALTAKAEEPAEAEGRVALM TLHNAKGLEFPVVELVGVEEGILPHRNSVETLEGLEEERRLFYVGITRAQERLYLSHAEE REVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERVVEPREGPG TVVAAQGDEVIVEFEGFGLKRLSLKYAELKPA >tr|B5YD55|B5YD55_DICT6 DNA helicase OS = Dictyogiomus thermophilum (strain ATCC 35947/DSM3960/H-6-12) GN = DICTH_0581 PE = 4 SV = 1 (SEQ ID NO: 100) MNNQFDSEKKIFIIPSRKKKEFLERIEKDLNEEQRKVVLEADGPSLVIAGPGSGKTRTIV YRVGYIVALGYSPKNIMLLTFTNQAARHMINRTQALIRESIEEIWGGIFEHVGNRILRVY GKIIGINEQYNILDREDSLDLIDECLEELFPEENLGKGILGELFSYKVNIGKNWDEVLKI KAPQIIDKIEIVQKVFERYEKRKRELNVLDYDDLLFTWYRLLLESEKTRKILNDRELYIL VDEYQDTNWLQGEIIRLTREENKNILVVGDDAQSIYSFRGATIENILSFPEIFPGTRIFy LVFNYRSTPEIINLANEIIKRNTRQYFKEIKPVLKSGSKPKLVWVRDDEEEAQFVVEVIK ELHKEGVKYKDIGVLERSNYHSMAVQMELTLQGIPYEVRGGLRFFEQAHIKDMISLLKIL FNEWEISAQRFFKLFPGIGRAYAKKLSQVLKESKDFDKIFQMUSGRTLEGLRILKNIW DKIKVIPVQNFSEILRVFFNEYYKDYLERNYPDFKDREKDVDQLILLSERYDDLEKFLSE LTLYTYAGEKLLEEEEEEKDFVVLSTIHQAKGLEWHAVFILRLVQGDEPSYKSMDNIEEE RRLFYVAVTRAKRELYVITYLTRKVKDMNVFTKPSIFLEELPYKELFEEWIVQREI >tr|F6DJA4|F6DJA4_THETG DNA helicase OS = Thermus thermophilus (strain SG0.5JP17-16) GN = Ththe16_124 PE = 4 SV = 1  (SEQ ID NO: 101) MLSPFGGEEETKAIPLEFEILLAWRVFSAALPPNFLAPVSASLHILVREAEGKEGAELEA YAWERLEELARTSVVKDAIQSFLEVAAEKPEVLRAGLLWFRTWNRLSPEEREALYRKAER FKPTAELASKASFLQGPPPPPKPLSPSVQAARSSPPRFTPTPEQEEAVRAFLSREDMKLV AVAGSGKITTLRLMAQSAPKERLLYVAFNRSVRDEAERTFPGNVEVLTLHGLAHRHVVRG SGAYQRKLAARNGRVTPGDV-LEALELPRERYALAYVIRSTLEAFLRSASEVPIPAHIPPE YREVLQRRDKDPFSERYVLKAVRLIWKLMQDPDDSFPLSEDGFVKIWAQAGAKIRGYDAY LVDEAQDLSPVFLQNLEAERGELRRVYVGDPRQQTYGWRGAVNAMDKLDAPERKLIWSFR FGEDLARGVRRFLAHVGSPIELHGKAPWDTEVSLARPEPPYTALCRTNAGAVEAVISFLL EEGREGARVEVVGGVDEIANLLRDAHLLKVGGEREKPHPELALVENWEELEELAKEVNHP QARMLVRLARRYDLLELARLLKHAQADEEGKADLVVSTLHKAKGREWDRVVLWGDFIPVW DEKVREFYRKQGALDELKEEENVVYVALTRARRFLGLDQLPDLHERFFQGEGLVKPPSVS PLSVGGAGVSADLLRELEVRVLAKLEDRLKEVAEVLAALLVEEASKAVAEAMREMGLLGE EG >tr|F6DIL2|F6DIL2_THETG DNA helicase OS = Thermus thermophilus (strain SG0.5JP17-16) GN = Ththe16 1438 PE = 4 SV = 1 (SEQ ID NO:  102) MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVERVAYLVARRGVEPSEILAVT FINKAAEEMRERLRGIVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFV-VIDEDDQTAL LKEV-LKELALSARPGPIKALLDRAKNRGVGLKALLGELPETYAGLSRGRLGDVLVRYQEA LKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFIRLLAGEE ANLMAVGDPDQGIYSFRADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKN ALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAOSRLL EQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATW ARVQLLAQEKGLPPWEALKEAARTFPRAEPLRHEVALVEELQDLVEGPAEAFFRHLLEAT DYPTYIREATPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGKVALM TLHNAKGLEFPVVELVGVEEGLLPHRNELSTLEGLEEERRLFYVGITRAQERLYLSHAEE REVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERVVHPREGPG TVVAAQGDEVTVHFEGVGLKRLSLKYAELKPA >tr|F6DJ67|F6DJ67_THETG DNA helicase OS = Thermus thermophilus (strain SG0.5JP17-16) GN = Tnthe16 2078 PE = 4 SV = 1(SEQ ID NO:  103) MEANLYVAGAGIGKTYTLAERYLGFLEEGLSPLQVVAVIFTERAALELRHRVRQMVGERS LGHKERVLAELEAAPIGTLHALAARVCREFPFEAGVPADFQVMEDLEAALLLEAWLEEAL LEALQDPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRALRR RMEELFHGERPEERYPGFPKGWRTEEPEVVPDLLAWAGEVKFNKKPWLEYKGDPALERLL KLLGGVKEGFSPGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRAL EREEVRAYYRGRFRRLLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQSIYSFRRADP KVMERTQAEAAKEGLRVRRLEKSHRYHQGLADEHNRFFPPLLPGYGAVSAERKPEGEGPW VEHFQGDLEAQARTIAQEVGRILSEGFQVYDLGEKAYRPMSLRDVAVLGRTWRDLARVAE ALRRLEVPAVEAGGGNLLETRAFKDAYLALRFLGDPKDEEALVGLLRSPFFALTDGEVRR LAEARGEGETLWEVLEREGDLSAEAERARETLRGLLRRKALEAPSRLLQRLDGATGYTGV AARLPQGRRRVKDWEGILDLVRKLEVGSEDPFLVARHLRLLLRSGLSVERPPLEAGEAVT LLTVHGAKGLEWPVVEVLNVGGWNRLGSWKNNKTKPLFRPGLALVPPVLDEEGNPSALFH LAKRRVEEEEKQEENRLLYVAATRASERLYLLLSPDLSPDKGDLDPQTLIGAGSLEKGLE ATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPLAARDPEAARRRLLGEPEPEGGEA WEPDGPQETEEEVPGGAGVGRMTHALLERFEAPEDLEREGRAFLEESFPGAEGEEVEEAL RLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVGEDWVLDYKTDRGVDAKA YLLQVGVYALALGKPRALVADLREGKLYEGASQWEEKAEEVLRRLMGGDRPEA >tr|G8N9P8|G8N9P8_9DEIN DNA helicase OS = Thermus sp. CCB US3 UF1 GN = TCCBUS3UF1 17030 PE = 4 SV = 1 (SEQ ID NO: 104) MDAFPSGKPLDEAWLSSLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLMARRGV YPSEILAVTFTNKAEEMRERLKAMVKGAGELWVSTFHAAALRILRFYGERVGLKPGFVV YDEDDQTALLKEVLKELGVSAKPGPIKALLDRAKNRGEPPERLLADLPEYYAGLSRGRLL D-VLHRYQQALWAQGALDFGDILLLALKLLEEDPEVRKRVRKRARFIHVDEYQDTSPVQYR LTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILUTEDFPGAKVIRLEENYRSTERILR FANAVIVKNALRLEKTLRPVKSGGEPVRLFRARDAREEAREVAEEVLRLGPPYDRVAVLY RTNAQSRILEQALASRGIGARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNIP PRGIGPATVEKVQAIAQEKGLPLYEALKVAAQVLPRPEPLRHFLALMEELMDLAFGPAEA FFRHLLEATDYPAYLKEAYPEDLEDRLENVEELLRAREAEGLMDFLDKVALTARAEEPG EAGGKVALMTLHNAKGLEFPVVFLVGVEEGLLPHRSSVSTLEGLEEERRLFYVGVTRAQE RLYLSYAEEREVYGRPEASRPSRFLEEVEEGLYEEYDPYRLPPPKPVPPPHRAKPGAFRG GEKVVHPRFGLGTVVAASGDEVIVHFDGVGLKRLSLKYADLRPA >tr|Q1J014|Q1J014_DEIGD DNA helicase OS = Deinococcus geothermalis (strain DSM11300) GN = Dgeo 0868 PE = 4 SV = 1  (SEQ ID NO: 105) MPDLPASSLLAQLNPNQAQAANHYTGPALVIAGAGSGKIRTLVYRIAHLIGHYGVDPGEI LAVTFINKAAAEMRERARHLVEGADRLWMSTEESAGVRILRAYGEHIGLKRGEVIYDDDD QLDILKEIMGSIPGIGAETHPRVLRGILDRAKSNLLTPADLARHPEPFISGLPREVAAEA YRRYEARKKGQNAIDEGDLITETVRLFQEVPAVLERVQDRARFIHVDEYQDTNKAQYELT RILASRDRNLLVVGDPDQSIYRFRGADIQNILDFQKDYLDAKVYMLEQNYRSSARVLTIA NKLIENNAERLEKTLRPVKEDGHPVLEHRATDQRAEGDEVAEWLIRLHAEGMRFSDMAVL YRTNAQSRVIEESLRRVQIPAKIVGGVGFYDRREIKDVLAYARLAINPDDDVALRRIIGR PKRGIGDTALERLMEWARVNGTSILTACAHAQELNILERGAQKAVEFAGLMHAMSEAADN DEPGPFLRYVIETSGYLDLLRQEGQEGQVRLENLEELVSAAEEWSRENEGTIGDFLDDAA LLSSVDDMRTKQENKDVPEDAVTLMTLHNAKGLEFPVVFIVGTEEGLLPSKNALLEPGGI EEERRLFYVGITRAMERLFLTAAQNRMQYGKTLATEDSRFLEEIKGGFDTVDAYGQVIDD RPKSWKEYRPTESARPGAVKNTSPLTEGMAYRGGEKVRHPKFGEGQVLAVAGLGDRQEVT VHFPSAGTKKLLVKFANLTRA 7>tr|Q745W4|045W4_THET2 DNA helicase OS = Thermus thermophilus (strain HB27/ATCC BAA-163/DSM7039) GN = TT P0191 PE = 4 SV = 1 (SEQ ID NO: 106) MALRPTEEQLKAVEAYRSGQDLKVVAVAGSGKITTLRIMAEATPGKRGLYLAFNRSVQQE AARKFPRNVRPYTLHALAFRMAVARDEGYRAKFQAGKGHLPAQAVAEALGLRNPLLLHAV LGTLEAFLRSEAASPDPGMIPLAYRTLRAGIKTWPEEEAFVLRGVEALWRRMTDPKDPFP LPHGAYVKLWALSEPDLSFAEALLVDEAQDLDPIFLKVLEAHRGRVQRVYVGDPRQQIYG WRGAINAMDRLEAPEARLTWSFRFAETLARFVRNLTALQDRPVEVRGKAPWATRVDAALP RPPFTVLCRTNAGVVGAVVVTHEVHRGRVEVVGGVEELVHLLRDAALLKKGEKRTDPHPD LAMVETWEELEALAEAGYAPAYGVLRLAQEHPDLEALAAYLERAWTPVEVAAGVVVSTAE KAKGREWDRVVLWDDFYPWWEEGWRVNWGSDPAHLEEENLLYVAATRARKHLSLAOIR DLLEAVDRMGVYRVAEEATRAYLLLSAEVLRGVATDPRVPAEHRVRALKALGYLERGEEA LDSPGKPGGQG >tr|Q721S0|021S0_THET2 DNA helicase OS = Thermus thermophilus (strain HB27/ATCC BAA-I63/DSM7039) GN = uvrD PE = 4 SV = 1  (SEQ ID NO: 107) MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVT FINKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTAL LKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVIVRYQEA LKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEE ANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKN ALRIEKALRPVKRGGEPVRIYRAEDAREFAREVAEEIARIGPPWDRYAVLYRTNAQSRLL EQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNIPPRGIGPATW ARVQLLAQEKGLPPWEALKEAARTFPRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEAT DYPAYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGRVALM TLHNAKGIEFPVVELVGVEEGLLPHRNEVSTLEGLEEERRLFYVGITRAQERLYLSHAEE REVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERVVHPRFGPG TVVAAQGDEVIVEFEGFGLKRLSLKYAELKPA >tr|F2NK78|F2NK78_MARHT DNA helicase OS = Marinithermus hydrothermalis (strain DSM14884/JCM11576/TI) GN = Marky_1312 PE = 4 SV = 1 (SEQ ID NO: 108) MDLLRDLNPAQREAVQHYTGPALVVAGAGSGKTRTVVERIAYLIRHRGVIPTEILAVTFT NKAAGEMKERLARMVGPAARELWVSTFHSAALRILRVIGEYIGLKPGFVVYDEDDQLALL KEVIGGLGLETRPQYARGVIDRIKNRMWSVDAFLREAEDWVGGLPKEQMAAVYQAYEARM RALGAVDMDTDLKVIGLFEABPEVLHRVQQRARFIHVDEYQDINPAQYRLTRLLAGAER NLMVVGDPDQSIYGFRNADIHNILNFEKDYPDARVYRLEENYRSTEAILRVANAVIERNA LRLEKTLRPVRSGGDPVFLYRAPDHREEAAFVAREVQRLKGRGRRLDEAVLYRTNAQSR VLEEAFRRQNLGVRIVGGVGFYERREVKDVLAYARAAVNPADDLAVKRVLNVPARGIGQT SLARDSQLAETARVSFFEALRRAGEVLARPQAQAVQRFVALIEGLANAAYDIGPDAFIRL VLAEIGYADMLRREPDGEARLENLEELLRAAREINEEQHAGIIADFLDEVALTARAEEPEG EVPAEAVILMILHNARGLEETVVFIVGVEEGLLPHRSSTARVEDLEEERRLFYVGIRAQ ERLYLILSEEREIYGRREAVRASREILEDIPEAFLQPLSPFGEPLGAGREPVAVRPTRRSS AAGGFRGGEKVRHPRFGQGLVVAASGEGDRQEVIVEIFAGVGLKKLLVKYAGLERIEL

REFERENCES

-   1. B. Choi, G. Zocchi, Y. Wu, S. Chan, L. Jeanne Perry, Allosteric     control through mechanical tension. Phys Rev Lett 95, 078102 (2005). -   2. M. Volgraf et al., Allosteric control of an ionotropic glutamate     receptor with an optical switch. Nat Chem Biol 2, 47-52 (2006). -   3. M. Tomishige, R. D. Vale, Controlling kinesin by reversible     disulfide cross-linking. Identifying the motility-producing     conformational change. J Cell Biol 151, 1081-1092 (2000). -   4. B. Schierling et al., Controlling the enzymatic activity of a     restriction enzyme by light. Proceedings of the National Academy of     Sciences of the United States of America 107, 1361-1366 (2010). -   5. D. M. Veine, K. Ohnishi, C. H. Williams, Jr., Thioredoxin     reductase from Escherichia coli: evidence of restriction to a single     conformation upon formation of a crosslink between engineered     cysteines. Protein science: a publication of the Protein Society 7,     369-375 (1998). -   6. T. M. Lohman, E. J. Tomko, C. G. Wu, Non-hexameric DNA helicases     and translocases: mechanisms and regulation. Nat Rev Mol Cell Biol     9, 391-401 (2008). -   7. A. Niedziela-Majka, M. A. Chesnik, E. J. Tomko, T. M. Lohman,     Bacillus stearothermophilus PcrA monomer is a single-stranded DNA     translocase but not a processive helicase in vitro. The Journal of     biological chemistry 282, 27076-27085 (2007). -   8. T. Ha et al., Initiation and re-initiation of DNA unwinding by     the Escherichia coli Rep helicase. Nature 419, 638-641 (2002). -   9. B. Sun et al., Impediment of E. coli UvrD by DNA-destabilizing     force reveals a strained-inchworm mechanism of DNA unwinding. The     EMBO journal, (2008). -   10. W. Cheng, J. Hsieh, K. M. Brendza, T. M. Lohman, E. coli Rep     oligomers are required to initiate DNA unwinding in vitro. Journal     of molecular biology 310, 327-350 (2001). -   11. N. K. Maluf, C. J. Fischer, T. M. Lohman, A Dimer of Escherichia     coli UvrD is the active form of the helicase in vitro. Journal of     molecular biology 325, 913-935 (2003). -   12. M. S. Dillingham, Superfamily I helicases as modular components     of DNA-processing machines. Biochemical Society transactions 39,     413-423 (2011). -   13. M. Yamaguchi, V. Dao, P. Modrich, MutS and MutL activate DNA     helicase II in a mismatch-dependent manner. The Journal of     biological chemistry 273, 9197-9201 (1998). -   14. L. E. Mechanic, B. A. Frankel, S. W. Matson, Escherichia coli     MutL loads DNA helicase II onto DNA. The Journal of biological     chemistry 275, 38337-38346 (2000). -   15. P. Soultanas et al., Plasmid replication initiator protein RepD     increases the processivity of PcrA DNA helicase. Nucleic acids     research 27, 1421-1428 (1999). -   16. A. F. Slatter, C. D. Thomas, M. R. Webb, PcrA helicase tightly     couples ATP hydrolysis to unwinding double-stranded DNA, modulated     by the initiator protein for plasmid replication, RepD. Biochemistry     48, 6326-6334 (2009). -   17. P. Soultanas, M. S. Dillingham, D. B. Wigley, Escherichia coli     ribosomal protein L3 stimulates the helicase activity of the     Bacillus stearothermophilus PcrA helicase. Nucleic acids research     26, 2374-2379 (1998). -   18. J. E. Yancey, S. W. Matson, The DNA unwinding reaction catalyzed     by Rep protein is facilitated by an RHSP-DNA interaction. Nucleic     acids research 19, 3943-3951 (1991). -   19. S. Barranco-Medina, R. Galletto, DNA binding induces     dimerization of Saccharomyces cerevisiae Pif1. Biochemistry 49,     8445-8454 (2010). -   20. H. S. Subramanya, L. E. Bird, J. A. Brannigan, D. B. Wigley,     Crystal structure of a DExx box DNA helicase. Nature 384, 379-383     (1996). -   21. S. Korolev, J. Hsieh, G. H. Gauss, T. M. Lohman, G. Waksman,     Major domain swiveling revealed by the crystal structures of     complexes of E. coli Rep helicase bound to single-stranded DNA and     ADP. Cell 90, 635-647 (1997). -   22. J. Y. Lee, W. Yang, UvrD helicase unwinds DNA one base pair at a     time by a two-part power stroke. Cell 127, 1349-1360 (2006). -   23. H. Jia et al., Rotations of the 2B sub-domain of E. coli UvrD     helicase/translocase coupled to nucleotide and DNA binding. Journal     of molecular biology 411, 633-648 (2011). -   24. K. M. Brendza et al., Autoinhibition of Escherichia coli Rep     monomer helicase activity by its 2B subdomain. Proceedings of the     National Academy of Sciences of the United States of America 102,     10076-10081 (2005). -   25. W. Cheng et al., The 2B domain of the Escherichia coli Rep     protein is not required for DNA helicase activity. Proceedings of     the National Academy of Sciences of the United States of America 99,     16006-16011 (2002). -   26. S. Myong, I. Rasnik, C. Joo, T. M. Lohman, T. Ha, Repetitive     shuttling of a motor protein on DNA. Nature 437, 1321-1325 (2005). -   27. G. Lee, M. A. Bratkowski, F. Ding, A. Ke, T. Ha, Elastic     Coupling Between RNA Degradation and Unwinding by an     Exoribonuclease. Science (New York, N.Y 336, 1726-1729 (2012). -   28. M. C. Murphy, I. Rasnik, W. Cheng, T. M. Lohman, T. Ha, Probing     single-stranded DNA conformational flexibility using fluorescence     spectroscopy. Biophysical journal 86, 2530-2537 (2004). -   29. J. A. Ali, T. M. Lohman, Kinetic measurement of the step size of     DNA unwinding by Escherichia coli UvrD helicase. Science (New York,     N.Y 275, 377-380 (1997). -   30. J. R. Moffitt et al., Intersubunit coordination in a homomeric     ring ATPase. Nature 457, 446-450 (2009). -   31. L. R. Brewer, P. R. Bianco, Laminar flow cells for     single-molecule studies of DNA-protein interactions. Nature methods     5, 517-525 (2008). -   32. T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles, S. M. Block,     Forward and reverse motion of single RecBCD molecules on DNA.     Biophysical journal 86, 1640-1648 (2004). -   33. J. G. Yodh, M. Schlierf, T. Ha, Insight into helicase mechanism     and function revealed through single-molecule approaches. Quarterly     reviews of biophysics 43, 185-217 (2010). -   34. W. Zhang et al., Directional loading and stimulation of PcrA     helicase by the replication initiator protein RepD. Journal of     molecular biology 371, 336-348 (2007). -   35. C. Machon et al., RepD-mediated recruitment of PcrA helicase at     the Staphylococcus aureus pC221 plasmid replication origin, oriD.     Nucleic acids research 38, 1874-1888 (2010). -   36. J. Park et al., PcrA helicase dismantles RecA filaments by     reeling in DNA in uniform steps. Cell 142, 544-555 (2010). -   37. M. J. Comstock, H. Jia, T. M. Lohman, T. Ha, Y. R. Chemla.     (2014). -   38. M. N. Dessinges, T. Lionnet, X. G. Xi, D. Bensimon, V.     Croquette, Single-molecule assay reveals strand switching and     enhanced processivity of UvrD. Proceedings of the National Academy     of Sciences of the United States of America 101, 6439-6444 (2004). -   39. J. G. Yodh, B. C. Stevens, R. Kanagaraj, P. Janscak, T. Ha, BLM     helicase measures DNA unwound before switching strands and hRPA     promotes unwinding reinitiation. The EMBO journal 28, 405-416     (2009). -   40. B. X. Huang, H. Y. Kim, Interdomain conformational changes in     Akt activation revealed by chemical cross-linking and tandem mass     spectrometry. Mol Cell Proteomics 5, 1045-1053 (2006). -   41. D. Branton et al., The potential and challenges of nanopore     sequencing. Nature biotechnology 26, 1146-1153 (2008). -   42. A. H. Laszlo et al., Decoding long nanopore sequencing reads of     natural DNA. Nature biotechnology, (2014). -   43. M. Vincent, Y. Xu, H. Kong, Helicase-dependent isothermal DNA     amplification. EMBO reports 5, 795-800 (2004). -   44. I. L. Urbatsch et al., Cysteines 431 and 1074 are responsible     for inhibitory disulfide cross-linking between the two     nucleotide-binding sites in human P-glycoprotein. The Journal of     biological chemistry 276, 26980-26987 (2001). -   45. I. Rasnik, S. Myong, W. Cheng, T. M. Lohman, T. Ha, DNA-binding     orientation and domain conformation of the E. coli rep helicase     monomer bound to a partial duplex junction: single-molecule studies     of fluorescently labeled enzymes. Journal of molecular biology 336,     395-408 (2004). -   46. R. Roy, S. Hohng, T. Ha, A practical guide to single-molecule     FRET. Nat Methods 5, 507-516 (2008). -   47. C. Joo, T. Ha, in Cold Spring Harb Protoc. (2012), vol. 2012. -   48. Y. Harada, K. Sakurada, T. Aoki, D. D. Thomas, T. Yanagida,     Mechanochemical coupling in actomyosin energy transduction studied     by in vitro movement assay. Journal of molecular biology 216, 49-68     (1990). -   49. T. Yanagida, M. Nakase, K. Nishiyama, F. Oosawa, Direct     observation of motion of single F-actin filaments in the presence of     myosin. Nature 307, 58-60 (1984). -   50. I. Rasnik, S. A. McKinney, T. Ha, Nonblinking and long-lasting     single-molecule fluorescence imaging. Nat Methods 3, 891-893 (2006). -   51. C. J. CLOPPER, E. S. PEARSON, THE USE OF CONFIDENCE OR FIDUCIAL     LIMITS ILLUSTRATED IN THE CASE OF THE BINOMIAL. Biometrika 26,     404-413 (1934). -   52. J. Gal, R. Schnell, S. Szekeres, M. Kalman, Directional cloning     of native PCR products with preformed sticky ends (autosticky PCR).     Molecular & general genetics: MGG 260, 569-573 (1999). -   53. Z. Qi, R. A. Pugh, M. Spies, Y. R. Chemla, Sequence-dependent     base pair stepping dynamics in XPD helicase unwinding. Elife     (Cambridge) 2, e00334 (2013). -   54. M. P. Landry, P. M. McCall, Z. Qi, Y. R. Chemla,     Characterization of photoactivated singlet oxygen damage in     single-molecule optical trap experiments. Biophysical journal 97,     2128-2136 (2009). -   55. C. Bustamante, Y. R. Chemla, J. R. Moffitt, High-resolution     dual-trap optical tweezers with differential detection.     Single-molecule techniques: a laboratory manual (Cold Spring Harbor     Laboratory Press, Cold Spring Harbor, N.Y., 2008). -   56. K. Berg-Sørensen, H. Flyvbjerg, Power spectrum analysis for     optical tweezers. Review of Scientific Instruments 75, 594-612     (2004).

All patents, patent applications, patent application publications and other publications that are cited herein are hereby incorporated by reference as if set forth in their entirety.

It should be understood that the methods, procedures, operations, composition, and systems illustrated in figures may be modified without departing from the spirit of the present disclosure. For example, these methods, procedures, operations, devices and systems may comprise more or fewer steps or components than appear herein, and these steps or components may be combined with one another, in part or in whole.

Furthermore, the present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various embodiments. Many modifications and variations can be made without departing from its scope and spirit. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art based on the foregoing descriptions. 

1. A modified helicase comprising a first subdomain having a first amino acid and a second subdomain having a second amino acid, wherein said first amino acid is at least about 30 Å from said second amino acid when the helicase is in an inactive conformation, and said first amino acid is less than about 20 Å from said second amino acid when the helicase is in an active conformation, and wherein a side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained helicase.
 2. The modified helicase of claim 1, optionally wherein: said modified helicase is a Super Family 1 (SF1) helicase or a Super Family 2 (SF2) helicase, optionally wherein said SF1 helicase is an SF1A or an SF1B helicase, and wherein if said modified helicase is SF1A or SF1B, the first subdomain optionally comprises a 1A subdomain or a 1B subdomain and the second subdomain optionally comprises a 2B subdomain; the first amino acid is less than about 15 Å, about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 5 Å, or about 4 Å from the second amino acid when the helicase is in an active conformation; the first amino acid is less than about 20 Å from the second amino acid when the helicase is in an active conformation; the first amino acid is at least about 50 Å, about 55 Å, about 60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å or about 85 Å from the second amino acid when the helicase is in an inactive conformation; the first amino acid is at least about 30 Å from the second amino acid when the helicase is in an inactive conformation; the first amino acid is at any one of positions 84-116 or 178-196 of the modified helicase amino acid sequence, and the helicase is selected from the group consisting of a Rep helicase, a PcrA helicase, and a UvrD helicase, or homolog thereof, optionally wherein: the first amino acid is at any one of positions 92-116 or 178-196 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof, optionally wherein the first amino acid is at position 187 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof; the first amino acid is at any one of positions 84-108 or 169-187 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof, optionally wherein the first amino acid is at position 178 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; or the first amino acid is at any one of positions 90-114 or 175-193 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof; the first amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:13 or SEQ ID NO:14, and the helicase is a Rep helicase, or homolog thereof; the first amino acid is at any one of positions 60-82 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; the first amino acid is at any one of positions 68-79 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; the first amino acid is at any one of positions 69-89 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof; the first amino acid is at any one of positions 77-87 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof; the first amino acid is at any one of positions 67-87 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof; or the first amino acid is at any one of positions 75-85 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof. 3-8. (canceled)
 9. The modified helicase of claim 1, optionally wherein said helicase is selected from the group consisting of a Rep helicase optionally from E. coli, a UvrD helicase optionally from E. coli, and a PcrA helicase optionally from B. stearothermophilus, optionally wherein: said helicase comprises one cysteine residue or no cysteine residues; or said helicase comprises one cysteine residue, optionally wherein said helicase is from a bacterium selected from the group consisting of Deinococcus geothermalis, Meiothermus sp., Marinithermus hydrothermalis, Oceanithermus profundus, Thermococcus sp. EXT9, Thermococcus sp. IRI48, Thermococcus sp. IRI33, Thermococcus sp. AMT7, Thermococcus nautili, Thermococcus onnurineus (strain NA1), Thermococcus kodakarensis (strain ATCC BAA-918/JCM 12380/KOD1) (Pyrococcus kodakaraensis (strain KOD1)), Thermococcus sibiricus (strain MM 739/DSM 12597), Thermococcus paralvinellae, Thermus aquaticus Y51MC23, Thermus aquaticus Y51MC23, Thermus aquaticus Y51MC23, Thermus sp. RL, Thermus sp. RL, Thermus sp. 2.9, Salinisphaera hydrothermalis C41B8, Thermus filiformis, Meiothermus ruber, Thermus sp. NMX2.A1, Thermus thermophilus JL-18, Thermus scotoductus (strain ATCC 700910/SA-01), Thermus scotoductus (strain ATCC 700910/SA-01), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKM B-2274/506), Thermus oshimai JL-2, Thermus oshimai JL-2, Thermus oshimai JL-2, Thermomonospora curvata (strain ATCC 19995/DSM 43183/JCM 3096/NCIMB 10081), Thermodesulfatator indicus (strain DSM 15286/JCM 11887/CIR29812), Geobacillus stearothermophilus (Bacillus stearothermophilus), Coprothermobacter proteolyticus (strain ATCC 35245/DSM 5265/BT), Meiothermus silvanus (strain ATCC 700542/DSM 9946/VI-R2) (Thermus silvanus), Anaerolinea thermophila (strain DSM 14523/JCM 11388/NBRC 100420/UNI-1), Thermoanaerobacterium thermosaccharolyticum M0795, Meiothermus ruber (strain ATCC 35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Meiothermus ruber (strain ATCC 35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Deinococcus radiodurans (strain ATCC 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/NCIMB 9279/R1/VKM B-1422), Thermodesulfobium narugense DSM 14796, Thermus thermophilus (strain HB8/ATCC 27634/DSM 579), Dictyoglomus thermophilum (strain ATCC 35947/DSM 3960/H-6-12), Thermus thermophilus (strain SG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermus sp. CCB US3 UF1, Deinococcus geothermalis (strain DSM 11300), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM 7039), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM 7039), and Marinithermus hydrothermalis (strain DSM 14884/JCM 11576/T1). 10-22. (canceled)
 23. The modified helicase of claim 1, optionally wherein: the second amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:15 or SEQ ID NO:16, and the helicase is a Rep helicase, or homolog thereof; wherein the second amino acid residue is at any one of positions 388-411, 422-444 and 518-540 of the modified helicase amino acid sequence, and the helicase is selected from the group consisting of a Rep helicase, a PcrA helicase, and a UvrD helicase, or homolog thereof, optionally wherein: the second amino acid is at any one of positions 397-411, 431-444 or 526-540 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof, optionally wherein the second amino acid is at position 409 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof; the second amino acid is at any one of positions 388-402, 422-435 or 519-531 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof, optionally wherein the second amino acid is at position 400 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; the second amino acid is at any one of positions 393-407, 427-440 or 523-540 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof; the second amino acid is at any one of positions 509-536 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; the second amino acid is at any one of positions 519-525 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; the second amino acid is at any one of positions 516-534 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof; the second amino acid is at any one of positions 526-532 of the modified helicase amino acid sequence, and the helicase is a PcrA helicase, or homolog thereof; the second amino acid is at any one of positions 513-531 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof; or the second amino acid is at any one of positions 523-529 of the modified helicase amino acid sequence, and the helicase is a UvrD helicase, or homolog thereof. 24-30. (canceled)
 31. The modified helicase of claim 1, optionally wherein: said first subdomain and said second subdomain comprise no more than a total of two cysteine residues; the first amino acid and the second amino acid are each independently an unnatural amino acid or a natural amino acid; one or more of an amino acid of the helicase is substituted with an unnatural amino acid or a natural amino acid, optionally wherein the amino acid is cysteine or homocysteine; and/or said helicase comprises a sequence selected from the group consisting of SEQ ID NOs:4 and
 12. 32-35. (canceled)
 36. The modified helicase of claim 1, optionally wherein: the first amino acid is covalently crosslinked to the second amino acid by a disulfide bond; and/or the first amino acid is covalently crosslinked to the second amino acid by a chemical crosslinker, optionally wherein: the chemical crosslinker is a bis-maleimide crosslinker; or the chemical crosslinker has a length of from about 6 Å to about 25 Å, optionally wherein the chemical crosslinker is selected from the group consisting of

1-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione,

1-[6-(2,5-dioxopyrrol-1-yl)hexyl]pyrrole-2,5-dione,

1-[2-[2-(2,5-dioxopyrrol-1-yl)ethyldisulfanyl]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)phenyl]pyrrole-2,5-dione,

N,N′-bis[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethyl]-N,N′-diphenylbutanediamide, and

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione. 37-42. (canceled)
 43. A modified Rep, PcrA or UvrD helicase or homolog thereof, comprising a first subdomain having a first amino acid at any one of positions 84-116 and a second subdomain having a second amino acid at any one of positions 388-411, 422-444 and 518-540, wherein a side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained Rep, PcrA or UvrD helicase, or homolog thereof.
 44. A modified Rep helicase or homolog thereof comprising an amino acid at position 178 covalently crosslinked to an amino acid at position 400 to form an active, conformationally-constrained Rep helicase or homolog thereof.
 45. A modified Rep helicase or homolog thereof comprising an amino acid at position 187 covalently crosslinked to an amino acid at position 409 to form an active, conformationally-constrained helicase.
 46. (canceled)
 47. A method of making an active, conformationally-constrained helicase comprising: selecting in a helicase a first amino acid in a first subdomain that is at least about 30 Å from a second amino acid in a second subdomain when the helicase is in an inactive conformation, and the first amino acid is less than about 20 Å from the second amino acid when the helicase is in an active conformation, and covalently crosslinking the first amino acid to the second amino acid when the helicase is in an active conformation to form an active, conformationally-constrained helicase.
 48. The method of claim 47, optionally wherein: said modified helicase is a Super Family 1 (SF1) helicase or a Super Family 2 (SF2) helicase, optionally wherein said SF1 helicase is an SF1A or an SF1B helicase, and wherein if said helicase is SF1A or SF1B, the first subdomain optionally comprises a 1A subdomain or a 1B subdomain and the second subdomain optionally comprises a 2B subdomain; the first amino acid is less than about 15 Å, about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 5 Å, or about 4 Å from the second amino acid when the helicase is in an active conformation; the first amino acid is at least about 30 Å, about 35 Å, about 40 Å, about 45 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å or about 85 Å from the second amino acid when the helicase is in an inactive conformation; said helicase is selected from the group consisting of a Rep helicase, a UvrD helicase and a PcrA helicase; or said helicase comprises a sequence selected from the group consisting of SEQ ID NOs:4 and
 12. 49-54. (canceled)
 55. The method of claim 47, wherein the first amino acid is covalently linked to the second amino acid by a disulfide bond, and/or the first amino acid is covalently linked to the second amino acid by a chemical crosslinker.
 56. (canceled)
 57. A method of catalyzing an unwinding reaction of a double-stranded DNA, comprising contacting the double-stranded DNA with the conformationally-constrained helicase of claim 1, optionally wherein: the conformationally-constrained helicase comprises SEQ ID NO:4 or SEQ ID NO:12; the conformationally-constrained helicase is chemically crosslinked; the linker comprises an alkyl having a length in the range from C7 to C23; or the linker comprises an alkyl having a length in the range from C8 to C13. 58-61. (canceled)
 62. A method of performing isothermal DNA amplification, comprising combining: a DNA template; the conformationally-constrained helicase of claim 1; and amplification reagents; under conditions compatible for performing isothermal DNA amplification.
 63. The method of claim 62, wherein: the conformationally-constrained helicase comprises SEQ ID NO:4 or 12; or the conformationally-constrained helicase is chemically crosslinked, optionally wherein the chemical crosslinker comprises a length in the range from about 6 Å to about 25 Å; the chemical crosslinker comprises an alkyl having a length in the range from C7 to C23; or the chemical crosslinker comprises an alkyl having a length in the range from C8 to C13.
 64. The method of claim 62, optionally further comprising the use of a DNA-dependent DNA polymerase selected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol I Large Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment, Bsu DNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase and phi29 DNA Polymerase. 65-68. (canceled)
 69. A kit for performing helicase dependent amplification, comprising: the conformationally-constrained helicase of claim 1; and amplification reagents, optionally further comprising a DNA-dependent DNA polymerase, optionally wherein the DNA-dependent DNA polymerase is selected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol I Large Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment, Bsu DNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase and phi29 DNA Polymerase.
 70. The kit of claim 69, wherein the conformationally-constrained helicase is selected from SEQ ID NOs: 4 and
 12. 71-72. (canceled)
 73. An isolated nucleic acid encoding the modified helicase of claim 1, optionally wherein said isolated nucleic acid is selected from the group consisting of SEQ ID NOs: 2, 3, 10 and
 11. 74. (canceled)
 75. A modified E coli. Rep helicase or homolog thereof comprising a first subdomain having a first amino acid, a second subdomain having a second amino acid, and an axis vector defined by the alpha carbon of ILE371 from which the vector originates and the alpha carbon of SER280 or the alpha carbon of ALA603, wherein theta is an angle of rotation of said first amino acid and said second amino acid around the axis vector, wherein a first theta between said first amino acid and said second amino acid is between about 60 degrees and about 155 degrees when the helicase is in an inactive conformation, and a second theta between said first amino acid and said second amino acid is between about 355 degrees and about 25 degrees when the helicase is in an active conformation, and wherein a side chain of the first amino acid is covalently crosslinked to a side chain of the second amino acid with a linker to form an active, conformationally-constrained helicase.
 76. The modified E coli. Rep helicase or homolog thereof of claim 75, optionally wherein: the first theta is about 133 degrees; the second theta is about 0 degrees; the axis vector is defined by the alpha carbon of ILE371 and the alpha carbon of SER280; the first amino acid is at any one of positions 84-108 or 169-187 of the modified helicase amino acid sequence; the first amino acid is at position 178 of the modified helicase amino acid sequence; the first amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:13 or SEQ ID NO:14; first amino acid is at any one of positions 60-82 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; or the first amino acid is at any one of positions 68-79 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof. 77-82. (canceled)
 83. The modified E coli. Rep helicase or homolog thereof of claim 75, optionally wherein: the second amino acid is at any one of positions 388-402, 422-435 or 519-531 of the modified helicase amino acid sequence; the second amino acid is at position 400 of the modified helicase amino acid sequence; the second amino acid is present in an amino acid sequence having at least 20% amino acid sequence identity to SEQ ID NO:15 or SEQ ID NO:16; the second amino acid is at any one of positions 509-536 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof; or the second amino acid is at any one of positions 519-525 of the modified helicase amino acid sequence, and the helicase is a Rep helicase, or homolog thereof. 84-102. (canceled) 